Long non-coding RNA (LncRNA) MNX1 antisense RNA 1(MNX1-AS1) is associated with the pathology of numerous cancers. But, the role and underlying pathways of MNX1-AS1 in the regulation of laryngeal squamous cell carcinoma (LSCC) is not known. We demonstrated remarkably elevated levels of MNX1-AS1 in the LSCC tissues, which was correlated with poor disease prognosis. Moreover, MNX1-AS1-silencing strongly suppressed LSCC cell proliferation, migration, and invasion. We also demonstrated that MNX1-AS1 sequesters that activity of miR-370, thereby releasing Forkhead Box ml (FoxM1) from the inhibitory actions of MNX1-AS1. Furthermore, the positive correlation of MNX1-AS1 and FoxM1 as well as the converse correlation between miR-370 and MNX1-AS1 (or FoxM1) were revealed in LSCC tissues using experiments. Based on rescue assays, FoxM1 overexpression or miR-370 downregulation partially recovered the inhibitory effect of MNX1-AS1 silencing on LSCC cells. Moreover, knockdown of MNX1-AS1 retarded tumor growth in nude mice model. In summary, these findings verified that MNX1-AS1 modulated LSCC progression by competitively binding with miR-370 to regulate FoxM1. Laryngeal squamous cell carcinoma (LSCC) is a highly prevalent form of laryngeal cancers that has been rising worldwide in recent years . AlthougLong non-coding RNAs (lncRNAs) are >200 nucleotides (nt) long RNAs that do not code for proteins and are associated with numerous forms of cancers via its regulation of chromatin remodeling, transcriptional modulation, and post-transcriptional control , 5. Multin vitro and in vivo experimentation, we revealed MNX1-AS1 silencing reduced LSCC growth and metastasis by targeting the miR-370/FoxM1 pathway. Hence, this study established a new regulatory pathway of MNX1-AS1/miR-370/FoxM1 axis that modulates LSCC progression.In this study, we demonstrated elevated MNX1-AS1 levels in LSCC tissues, which closely related with advanced UICC stage, lymph node metastasis, and poor prognosis. Additionally, using both To establish MNX1-AS1 levels in LSCC, we examined MNX1-AS1 levels in LSCC tissues and adjoining healthy tissue (ANT) samples. As illustrated in MNX1-AS1 levels were assessed in a LSCC cell line TU212 and in normal bronchial epithelial cell line (16HBE). As depicted in Using wound healing and transwell invasion assays, we measured the impact of MNX1-AS1 depletion on LSCC migration and invasion abilities. We observed that MNX1-AS1 depletion suppressed TU212 cell migration and invasion  and 3B.https://bigd.big.ac.cn/lncbook/index). Based on the bioinformatics analysis, we discovered that MNX1-AS1 shares sequence homology with miR-370 (Cytoplasmic lncRNAs exerted their function through serving as competing endogenous RNAs (ceRNAs) to sequester miRNAs , 23. To  miR-370 . Lucifer miR-370 . To furt miR-370 . Moreove miR-370 , whereas miR-370 . Additio miR-370  and 4H.  miR-370 . Based oA previous study demonstrated that miR-370 inhibited LSCC tumorigenesis and development by targeting FoxM1 . To asseMultiple reports confirmed that lncRNAs played a vital role in LSCC cell physiology and pathogenesis, and might act as diagnosis markers and therapy agents , 7. Herein vitro, as well as inhibited tumorigenesis of LSCC in vivo. These data imply that MNX1-AS1 serves as an oncogene in LSCC.MNX1-AS1 was reported to serve as an oncogenic lncRNA in numerous forms of cancers –20. For A great evidence suggested that lncRNA can sequester miRNA and halt their suppression of miRNA-target genes . MNX1-ASForkhead box M1 (FoxM1) is a well established downstream target of miR-370 in LSCC . FoxM1, In summary, we showed that MNX1-AS1 depletion retarded LSCC progression via miR-370/FoxM1 axis. Thus, MNX1-AS1 may be a promising new therapeutic target for LSCC. However, further studies are required to provide a deep understanding of the clinical translation of the MNX1-AS1/miR-370/FoxM1 axis in LSCC.40 LSCC tissue samples and adjoining non-cancerous matched tissue samples were retrieved from patients undergoing partial or total laryngectomy between March 2014 and Mach 2015 at the First Hospital of Jilin University, under approval from Jilin University. All informed consents were signed by all patients. All patients did not receive any tumor treatment before admission. All tissues were flash-frozen in liquid nitrogen within 10 min of extraction and stored at −80°C until further examination.ΔΔCt method. Internal controls were GAPDH and U6 for MNX1-AS1/FOXM1 and miR-370, respectively.Total RNA was extracted from cells or tissues with TRIzol reagent  following manufacturer’s guidelines. The RNA was employed for the synthesis of cDNAs with Primescript RT reagent kit . cDNAs were amplified and quantified by with SYBR Green mix (Takara) in Applied Biosystems 7500 instrument. Primer sequences used in this study were described previously , 24, 28.Human LSCC cell line TU-212 and healthy bronchial epithelial cell line (16HBE) were bought from Shanghai Huiying Biological Technology , and maintained in Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin in a humid environment at 37°C with 5% CO2.® 3000 (Invitrogen), following manufacturer’s guidelines. 2 × 105 TU212 cells were plated and incorporated with pLKO.1-puro-sh-MNX1-AS1 or pLKO.1-puro-sh-NC for 72 h. Stable cell lines were chosen with 0.5 μg/ml puromycin and 250 μg/ml G418 for one week.The plasmids used were as follows: shRNA against MNX1-AS1 (sh-MNX1-AS1) and negative control (NC) shRNA (sh-NC) in pLKO.1-puro vector ; miR-370 mimics, miR-370 inhibitor (anti-miR-370) and their NC (miR-NC or anti-miR-NC) . TU212 cells were incorporated with the indicated plasmid, namely, either mimic, inhibitor or shRNA with Lipofectamine3 cells/well) for 24, 48 or 72 h. Next, CCK8 solution was introduced followed by a 4-h incubation. Absorbance was examined at 450 nm with a Microplate Reader .The impact of MNX1-AS1 on proliferation assay was evaluated using the Cell Counting Assay kit , based on manufacturer’s guidelines. Transfected cells were seeded in 96-well plates . For transwell invasion assay, 24-well Transwell chambers with polycarbonate filters  were applied. Plasmid incorporated cells (1×104 cells per well) in zero serum medium were introduced to the top chamber coated with Matrigel, and medium with 10% FBS was included in the bottom chamber for chemoattraction. After 24-h, the migrated cells were fixed with 4% formaldehyde for 20 min and then stained with 0.1% crystal violet for 5 min. Cell invasion was quantified with an inverted microscope  by choosing 5 random vision field per treatment.Wound healing and transwell chamber assays were carried out to assess migration and invasion properties, as we previously described (9). For wound healing assay, 2 × 10The NE-PER Nuclear and Cytoplasmic Extraction Reagents  were used to isolate the cytoplasmic and nuclear extracts from TU212 cells. The distribution of MNX1-AS1 in cytoplasm or nucleus was examined using qRT-PCR. GAPDH and U6 served as controls for the cytoplasm and nucleus, respectively.A wild-type (WT) or mutant (MT) MNX1-AS1 fragment containing the miR-370 binding site were introduced into the pGL3-basic vector .For luciferase assay, TU212 cells were simultaneously incorporated with the miR-370 mimics or corresponding NC. 48 h later, luciferase reporter assay system (Promega) was applied to examine the luciferase activity.TU212 cells were incorporated with biotinylated wide-type miR-370(Bio-wt-miR-370), mutant-type miR-370 (Bio-mt-miR-370) and negative control , respectively. After 48 h transfection, lysed cells were harvested and exposed to M-280 streptaviden magnetic beads (Invitrogen) based on manufacture’s guidelines. The bead-bound-MNX1-AS1 was then quantified using qRT-PCR.ad libitum. TU212 cells stably incorporated with sh-MNX1-AS1 or sh-NC were subcutaneously administered into flank of nude mice (Five mice in each group). Tumor volumes were quantified weekly formulas follows: tumor volume (V) = width2 × length × 0.5. After 28 days, all mice were sacrificed, tumors were harvested, and weighted. The tumor was paraffin-embedded to examine the cell proliferation marker Ki-67 expression with immunohistochemical (IHC) using an anti-Ki-67 antibody  as described previously [All animal protocols in this study were agreed upon by the Animal Research Ethics Committee of Jilin University . Ten male athymic nude BALB/c mice  from the Laboratory Animal center of Jilin University were housed in this center. All mice received standard mouse irradiated food and tap water eviously . The rest-test (2 groups data) or one-way analysis of variance (>2 groups data). A χ2 test was used for comparison of dichotomous variables. The differences in overall survival rate was assessed using Kaplan-Meier method and analyzed with log-rank test. Pearson's correlation analysis was employed for relationship investigations. A P value < 0.05 was considered as significant difference.Each experiment was repeated 3 times and analyzed with SPSS software, version 17.0 . The data presented is average ± SD (standard deviation). Continuous variables were analyzed with Student’s two-tailed


Higher valency pneumococcal conjugate vaccines (PCV10 and PCV13) replaced PCV7, and an updated global analysis of PCV impact on invasive pneumococcal disease (IPD) incidence is needed. We aimed to estimate the change in vaccine-type (VT), non-VT type and all-serotype (ST) IPD incidence following introduction of PCV10/13 among children < 5 years of age.IPD ST-specific incidence or cases and population denominators were obtained directly from surveillance sites. IPD incidence rate ratios (IRRs) for each site were estimated comparing the pre-any PCV incidence to each post-PCV10/13 year using Bayesian multi-level, mixed effects Poisson regressions. All-site weighted average IRRs were estimated using linear mixed-effects regressions. Results were stratified by product (PCV10 vs. PCV13) and years of prior PCV7 use .Analyses included 45 surveillance sites from 31 countries, primarily high-income (80%). Thirty surveillance sites had pre- and post-PCV data . Five years after PCV10/13 introduction, the all-site IRRs in children < 5 years were generally similar across products and prior PCV7 use strata for all-serotype IPD (range 0.23-0.41), PCV7 STs (0.01-0.13), PCV10non7 STs , and ST6A (0.01-0.18). IRRs for ST19A were lower for PCV13 sites (range by PCV7 use: 0.09-0.31) than for PCV10 sites (1.1-1.4). ST3 IRRs were dynamic, differing by product at year 5 , but converging by year 7. NonPCV13 STs increased across all strata (range 1.9-2.6), except one strata with a single African site that declined.Figure 1. All-Site Weighted Average Incidence Rate Ratios, Children <5>* Total sites indicates number of sites with incidence rate data included and pre/post sites indicates number of sites with both pre- and post-PCV data to estimate IRRs for each outcome. ** Year 0 indicates the year of PCV10/13 introduction and year -1 indicates the last year of PCV7 use prior to PCV10/13 introduction.All-serotype IPD in children < 5 years declined following both PCV10 and PCV13 use, driven by substantial declines in VT serotypes and offset by increases in nonPCV13 STs. ST19A decreased among PCV13-sites, mitigating replacement disease occurring after PCV7 use, but increased, on average, among PCV10-sites. Changes in ST3 were heterogeneous, increasing in some sites and no change from baseline in others. Data from low-income and high-burden settings were limited.Julia C. Bennett, MSPH, Pfizer (Research Grant or Support) Maria Deloria Knoll, PhD, Merck (Research Grant or Support)Pfizer (Research Grant or Support)


The attenuated total reflection-Fourier transform infrared (ATR-FTIR), scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDX) mapping analysis, and thermal analysis (TGA-DTA) were used to characterize the synthesized UiO-66-NH2 nanoparticles. The morphology, surface chemistry, and the gas separation performance of the fabricated Pebax® 2533-UiO-66-NH2/PP mixed matrix thin film hollow fiber membranes were characterized by using SEM, ATR-FTIR, and gas permeance measurements, respectively. It was found that the surface morphology of the prepared membranes was influenced by the incorporation of UiO-66 nanoparticles. The CO2 permeance increased along with an increase of UiO-66 nanoparticles content in the prepared membranes, while the CO2/N2 ideal gas selectively firstly increased then decreased due to the aggregation of UiO-66 nanoparticles. The Pebax® 2533-UiO-66-NH2/PP mixed matrix thin film hollow fiber membranes containing 10 wt% UiO-66 nanoparticles exhibited the CO2 permeance of 26 GPU and CO2/N2 selectivity of 37.Membrane separation technology can used to capture carbon dioxide from flue gas. However, plenty of research has been focused on the flat sheet mixed matrix membrane rather than the mixed matrix thin film hollow fiber membranes. In this work, mixed matrix thin film hollow fiber membranes were fabricated by incorporating amine functionalized UiO-66 nanoparticles into the Pebax The lower coating solution concentration could result in smaller selective layer thickness. The smooth PTMSP gutter layer can prevent the intrusion of Pebax® into pores of support layer, resulting in a thin selective layer. As a result, the prepared UiO-66-NH2/Pebax® 1657 based hollow fiber composite membranes showed very high gas separation performance. As it is shown in 2/N2 selectivity but much higher CO2 permeance than that of dense flat sheet mixed matrix membranes. Therefore, the gas separation performances of thin film mixed matrix membranes are better than the dense flat sheet membranes.The performance of the prepared Pebax fillers . The gasterature ,50,51,52® 2533-UiO-66-NH2/PP mixed matrix thin film hollow fiber membranes were successfully fabricated by using dip coating method. The pre-treatment of PP hollow fibers by dip-coating with 3 wt% Pebax® 2533 solution could smoothen the outer surface of hollow fiber supports, which facilitated the formation of defect-free selective layer. The incorporation of UiO-66-NH2 nanoparticles into the Pebax® 2533 coating solution affected the morphology, surface chemistry, and gas separation performance of Pebax® 2533-UiO-66-NH2/PP mixed matrix thin film hollow fiber membranes confirmed by SEM analysis, FTIR analysis, and gas permeance measurements, respectively. The aggregation of UiO-66-NH2 nanoparticles was observed at higher amounts of UiO-66-NH2 nanoparticles in the Pebax® 2533 matrix. The filler aggregation should be tackled by post-synthetic modification of UiO-66-NH2 nanoparticles. The CO2 permeance increased with the increase of the loading amount of UiO-66 nanoparticles, while the CO2/N2 ideal gas selectively firstly increased then decreased due to the aggregation of UiO-66 nanoparticles. The Pebax® 2533-UiO-66-NH2/PP mixed matrix thin film hollow fiber membranes containing 10 wt% UiO-66 nanoparticles exhibited the best gas separation performance with CO2 permeance of 26 GPU and CO2/N2 selectivity of 37.Pebax


These data demonstrate that fully vaccinated individuals developed robust anamnestic antibody and T cell responses following infection with the SARS-CoV-2 delta variant. Moreover, these findings suggest that population immunity will likely increase over time by a combination of widespread vaccination and breakthrough infections.Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been reported frequently in vaccinated individuals with waning immunity. In particular, a cluster of over 1,000 infections with the SARS-CoV-2 delta variant was identified in a predominantly fully vaccinated population in Provincetown, Massachusetts in July 2021. In this study, vaccinated individuals who tested positive for SARS-CoV-2 (n=16) demonstrated substantially higher serum antibody responses than vaccinated individuals who tested negative for SARS-CoV-2 (n=23), including 32-fold higher binding antibody titers and 31-fold higher neutralizing antibody titers against the SARS-CoV-2 delta variant. Vaccinated individuals who tested positive also showed higher mucosal antibody responses in nasal secretions and higher Spike protein-specific CD8 SARS-CoV-2 breakthrough infections led to robust serum and mucosal antibody responses and peripheral T cell responses in vaccinated individuals. A large cluster of coronavirus disease 2019 (COVID-19) infections was reported to the Massachusetts Department of Public Health (MA DPH) after the July 4, 2021 weekend in Provincetown, Barnstable County, Massachusetts  in Boston, Massachusetts. We measured peripheral antibody responses, mucosal antibody responses, and cellular immune responses in this cohort of vaccinated individuals with or without SARS-CoV-2 breakthrough infection.table S1). Participants were predominantly white, and the majority were male. Vaccinated infected individuals were generally younger  than vaccinated uninfected individuals . Participants received the BNT162b2 , mRNA-1273 , or Ad26.COV2.S  vaccines. Peripheral blood and nasal swab samples were obtained for immunologic assays at a median of 189 days (range 127 to 251) following first vaccine dose and a median of 34 days (range 0 to 67) following nasopharyngeal SARS-CoV-2 polymerase chain reaction (PCR) testing in the outbreak investigation. In the vaccinated infected group, 15 of 16 (94%) of individuals reported symptoms of COVID-19 infection, most commonly respiratory symptoms, fever, and loss of smell or taste, consistent with the overall outbreak investigation (table S2).A total of 39 individuals from the MA DPH outbreak investigation . RBD-specific ELISA IgG titers were 46-fold greater in vaccinated infected participants compared with unvaccinated infected participants .SARS-CoV-2 antibody responses in serum were assessed by Spike protein- and Nucleocapsid-specific electrochemiluminescence assays (ECLAs), receptor binding domain (RBD)-specific IgG enzyme-linked immunosorbent assays (ELISAs), and pseudovirus neutralizing antibody assays. Spike protein-specific ECLA titers against the SARS-CoV-2 WA1/2020, B.1.1.7 , B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.617.1 (kappa) variants were 14-, 15-, 11-, 18-, 15-, and 22-fold higher, respectively, in vaccinated infected individuals compared with vaccinated uninfected individuals (fig. S2). Among vaccinated uninfected individuals, binding and neutralizing antibody responses induced by mRNA-1273 were higher than BNT162b2, and individuals who received BNT162b2 generally had low or undetectable neutralizing antibody responses against the SARS-CoV-2 delta variant at the timepoint analyzed, consistent with waning immunity (fig. S2C). No correlations were observed between participant age and the titers of binding and neutralizing antibodies in this cohort (fig. S3). Moreover, robust antibody titers were observed in vaccinated infected individuals regardless of time from vaccination (fig. S4). Taken together, these data demonstrate markedly higher binding and neutralizing antibody responses in vaccinated individuals who tested positive for SARS-CoV-2 after vaccination compared with vaccinated individuals who tested negative for SARS-CoV-2.Similar trends were observed in subgroup analyses of participants who received BNT162b2 or mRNA-1273 (+ T cell responses to SARS-CoV-2 WA1/2020 and B.1.617.2 (delta) were 2.9- and 1.6-fold higher, respectively, in vaccinated infected individuals compared with vaccinated uninfected individuals (+ T cell responses to SARS-CoV-2 WA1/2020 and B.1.617.2 (delta) were below the limit of detection in vaccinated uninfected individuals but were at least 4.9- and 5.7-fold higher, respectively, in vaccinated infected individuals .SARS-CoV-2 cellular immune responses were assessed by intracellular cytokine staining assays and ELISPOT assays using pooled Spike peptides. Spike protein-specific CD4Antibody responses in the nasal mucosa may be critical for protection against respiratory viruses. In a subset of 23 individuals from whom nasal swabs were available, we assessed RBD-specific IgA and IgG responses in mucosal secretions extracted from nasal swabs. Nasal RBD-specific IgA responses to SARS-CoV-2 WA1/2020 and B.1.617.2 (delta) were 4-fold higher in vaccinated infected individuals compared with vaccinated uninfected individuals  study team for recruitment and informed consent. Participants also self-referred from flyers posted on social media. Participants were asked to provide their vaccine, symptom, and testing history, as well as their race and ethnicity based on specified categories; they could select multiple race categories. The BIDMC Institutional Review Board (IRB) approved this study (#2021P000344) as part of a parent biorepository study (#2020P000361). All participants provided informed consent. Breakthrough SARS-CoV-2 infection was defined as a positive nasopharyngeal swab PCR test after being fully vaccinated (at least 2 weeks following two doses of the BNT162b2 or mRNA-1273 vaccines or at least 2 weeks after a single dose of the Ad26.COV2.S vaccine). Participants were asked to provide blood samples and nasal swabs. Participants were excluded if they had past infection with SARS-CoV-2 prior to vaccination or received an additional booster dose of vaccine during the study period. Participants were enrolled as a convenience sample. No a priori power analysis was performed and no outliers were excluded. We also obtained samples (n=18) from unvaccinated participants in Boston with SARS-CoV-2 infection between March to November 2020 as part of the parent biorepository study (#2020P000361). The majority of these participants had asymptomatic or mild illness . Assay operators were all blinded to the participant prior infection or vaccination history.ECLA plates  were designed and produced with up to 9 antigen spots in each well, and assays were performed essentially as described previously  according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction (NT50) in relative light units was observed relative to the average of the virus control wells. Serum samples with dense lipophilic debris after heat inactivation were excluded from the pseudovirus neutralization analysis due to the inability to acquire an accurate measure of relative light units.SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were used to measure pseudovirus neutralizing antibodies as described previously  supplemented with CD49d monoclonal antibody  and CD28 monoclonal antibody  as described previously  were prepared and plated at a concentration of 2 μg/well, and 100,000 cells per well were added to the plate. The peptides and cells were incubated for 15 to 20 hours at 37°C. All steps following this incubation were performed at room temperature. The plates were washed with ELISPOT wash buffer and incubated for 2 to 4 hours with biotinylated mouse anti-human IFN-γ monoclonal antibody from MabTech (1 μg/mL). The plates were washed a second time and incubated for 2 to 3 hours with conjugated goat anti-biotin alkaline phosphatase from Rockland, Inc. (1.33 μg/mL). The final wash was followed by the addition of Nitro-blue Tetrazolium Chloride/5-bromo-4-chloro 3 ‘indolyphosphate p-toludine salt (NBT/BCIP chromagen) substrate solution for 7 min. The chromagen was discarded and the plates were washed with water and dried in a dim place for 24 hours. Plates were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.Descriptive statistics were calculated using GraphPad Prism 8.4.3. All data points represent biological replicates for each group. Each immunologic assay was performed for each sample independently without technical replicates. Data are presented as median with interquartile range (IQR). Comparisons were performed using the Wilcoxon rank-sum test. Spearman correlation coefficient (r) was reported for antibody titers with time from first vaccine dose and age. All tests were two-tailed. When all six variants were compared between infected and uninfected or between BNT162b2 and mRNA-1273, a p value < 0.01 was considered statistically significant to account for multiple comparisons. For all other comparisons, p values < 0.05 were considered statistically significant.


Although nucleic acid medicines are expected to function as new therapeutic agents, their targeted delivery into cancer cells, particularly hematologic cancer cells, via systemic administration, is limited. Based on our previous finding that tumor cell-derived exosomes are preferentially incorporated into their parental cancer cells, we previously demonstrated that anti-CD63 monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers (9mer) were transferred into solid cancer cells and inhibited exosomal miRNA functions. To challenge the delivery of siRNAs into hematologic cancer cells, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNA) with branched Arg linkers (9+9mer). Anti-CD63 mAb-conjugated complexes were incorporated into multiple myeloma (MM) cells. Moreover, these exosome-capturing mAb-conjugated siRNAs successfully decreased the mRNA transcript levels of targeted mRNAs in the MM cells. This technology could lead to a breakthrough in drug delivery systems for hematologic cancer therapy.luciferase+ (luc+) siRNAs significantly decreased the luminescence intensity in OPM-2-luc+ cells. Moreover, treatment with anti-CD63 mAb-conjugated with MYC and CTNNB1 siRNAs decreased the mRNA transcript levels of MYC and CTNNB1 to 52.5% and 55.3%, respectively, in OPM-2 cells. In conclusion, exosome-capturing Ab-conjugated siRNAs with branched Arg linkers can be effectively delivered into MM cells via uptake of exosomes by parental cells. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic cancers.Nucleic acid medicines have been developed as new therapeutic agents against various diseases; however, targeted delivery of these reagents into cancer cells, particularly hematologic cancer cells, via systemic administration is limited by the lack of efficient and cell-specific delivery systems. We previously demonstrated that monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers were transferred into solid cancer cells and inhibited exosomal miRNA functions. In this study, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNAs) with branched Arg linkers and investigated their effects on multiple myeloma (MM) cells. Anti-CD63 mAb-conjugated siRNAs were successfully incorporated into MM cells. The incorporation of exosomes was inhibited by endocytosis inhibitors. We also conducted a functional analysis of anti-CD63 mAb-conjugated siRNAs. Ab-conjugated  Nucleic acid medicines including small interfering RNAs (siRNAs), antisense oligonucleotides, and RNA aptamers have been developed as new therapeutic agents that directly suppress target molecules and control the pathophysiology of many diseases ,2,3,4. AMultiple myeloma (MM) is a hematologic malignancy characterized by malignant plasma cell proliferation, production of M-proteins, and associated with organ damage, including bone lesion, renal failure, anemia, and hypercalcemia . MM remaExosomes are extracellular vesicles of diameter 50–100 nm, which contain nucleic acids, lipids, and proteins, and control intercellular communication in both healthy physiology and pathophysiology ,13. In rlucifease+ (luc+) cells  vector (luc2 reporter gene 499–2151)  using Lipofectamine 3000 , according to the manufacturer’s instructions.To confirm the function of Ab-conjugated complexes, we established HeLa-luc cells. Approximately 1 × 109   or Cys(Npys)-(D-Arginine)9+(D-Arginine)9  . After completion of the reaction, gel filtration was performed using Zeba Spin Desalting Columns (7K MWCO) (Thermo Fisher Scientific) to remove unreacted Traut’s Reagent. Thereafter, the anti-CD63 mAb-linear (9mer) or branched (9+9mer) Arg was mixed with siRNAs at the indicated molar ratios and incubated at room temperature for 15 min to obtain anti-CD63 mAb-conjugated siRNA complexes. The scheme for the synthesis of anti-CD63 mAb drug conjugate with a polyarginine linker is shown in Anti-CD63 mAb  was used to develop Ab-conjugated siRNA complexes to capture exosomes derived from MM cells. To synthesize antibody-drug conjugates (ADCs), anti-CD63 mAb was bound to siRNA molecules, as previously described . In brieluc2 (for HeLa-luc cells) and siRNA against luc+ (for OPM-luc+ cells). FITC-labeled luc2-siRNA (F-luc2 siRNA) was used to investigate the cellular uptake of Ab-conjugated complexes by laser scanning microscopy. SiRNAs against MYC and CTTNB1 were synthesized to evaluate the suppression of targeted mRNAs in MM cells using quantitative real-time PCR (qPCR), as both molecules have essential roles in MM pathogenesis [MYC (Silencer® Select siRNA s9129 and s9130) were purchased from Thermo Fisher Scientific, and both siRNA #1 and siRNA #2 against CTTNB1 were designed and synthesized . SiRNA sequences are presented in Two siRNAs were synthesized to conduct luciferase assays to assess ant-CD63 mAb-conjugated siRNA function: siRNA against ogenesis ,23 and aogenesis ,25,26. S3/well in a 96-well plate. The next day, we generated ADCs by mixing anti-CD63 mAb combined with linear Arg linker and either Silencer Select negative control siRNA #2 (Thermo Fisher Scientific) or luc-2 siRNA at a ratio of 2.5:1 (750:300 (nM)). Twenty-four hours after treatment with ADCs, cells were transfected with the pGL4 luciferase reporter vector  using Lipofectamine 3000 (Thermo Fisher Scientific) and then luminescence was measured using a microplate reader . Efficacy was also evaluated in OPM-2-luc+ cells. OPM-2-luc+ cells were seeded at 1.5 × 103/well in a 96-well plate, and then treated with ADCs generated by mixing anti-CD63 mAb combined with linear Arg linker and Silencer Select negative control siRNA #2 (Thermo Fisher Scientific) or luc+ siRNA at a ratio of 1:1 (1500:1500 (nM)). Luminescence activity was measured using a microplate reader , according to the manufacturer’s instructions.We first evaluated the efficacy of anti-CD63 mAb-conjugated siRNA complexes using HeLa cells seeded at 4.5 × 103/well. Then, cells were treated with ADC anti-CD63 Abs with linker:F-luc2 siRNA at the indicated ratios. Twenty-four hours after ADC treatment, cells in glass-bottomed dishes were fixed with 4% paraformaldehyde , then treated with rhodamine-phalloidin  and Hoechst 33342  for 20 min and 5 min, respectively, at room temperature in the dark. Fluorescence images were acquired on an LSM800 laser confocal microscope (LSM) .OPM-2-luc+ cells and NCI-H929 cells were seeded in glass-bottomed dishes  coated with fibronectin solution  at 1.5 × 104/well. After 24 h incubation, cells were transferred to glass-bottomed dishes and then treated with endocytosis inhibitors, Rottlerin [luc2 siRNA labeled with FITC. Twenty-four hours after ADC treatment, OPM-2-luc cells were stained with Hoechst 33342 (5 μM) for 10 min at room temperature in the dark. Fluorescence images were acquired on an LSM800 laser confocal microscope (Carl Zeiss).The effects of inhibition of micropinocytosis using endocytosis inhibitors on anti-CD63 mAb-conjugated siRNA complex uptake into MM cells were investigated. OPM-2-luc cells were seeded in a 96-well plate at 1.5 × 10ottlerin   mRNA, OPM-2-luc+ cells were cultured in a 96-well plate at 1.5 × 104/well. After 24 h incubation, ADC comprising anti-CD63 mAb-branched Arg (1500 nM) and siRNA (1500 nM) was added. After 48 h of ADC treatment, RNA was purified using the NucleoSpin RNA Kit  and subjected to reverse transcription using a ReverTra Ace® qPCR RT Kit . For targeting c-MYC, OPM-2-luc+ cells were seeded in a 96-well plate at 7.5 × 103/well. After 24 h incubation, ADC comprising anti-CD63 mAb-branched Arg (1500 nM) and siRNA (1500 nM) was added. After 96 h treatment, RNA purification and reverse transcription were performed as described above. Human CTNNB1, MYC, and 18S ribosomal RNA (18S rRNA) mRNA expression levels were measured by real-time PCR. Each real-time PCR reaction mixture contained 20 μL of TaqMan master mix , cDNA, a primer pair, and a TaqMan probe . cDNA samples were amplified using a Thermal Cycler Dice system  with the following parameters: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. For normalization of loading differences, 18S rRNA mRNA was used as an internal control. The primers and TaqMan probes used in this study are shown in To investigate the efficacies of Ab-conjugated siRNA complexes in suppressing targeted mRNA transcripts in MM cells, two targets, β-catenin ,26 and ct-test. A p-value < 0.05 was considered statistically significant.For luciferase assays, comparisons between two groups were conducted using the unpaired We first developed two types of Arg linkers: linear-  and branched-  chain polyarginine; gel shift assays were conducted using native polyacrylamide gel electrophoresis to determine the optimal ratios of anti-CD63 mAb-to-siRNA. As shown in We investigated the incorporation of Ab-conjugated siRNA complexes into MM cells by examination using an LSM. First, we determined whether ADCs could transfer into adherent HeLa cells. Anti-CD63 mAb-linear Arg conjugated siRNAs was treated at a mAb-to-siRNA ratio of 5:1 (1500:300 (nM)). Confocal images showed that anti-CD63 Ab-linear Arg (9mer) conjugated with siRNAs was successfully transferred into HeLa cells a, and 3Dluc2 siRNA ratio of 5:1. Compared with a transfer of FITC-labeled siRNAs bound to ADCs with linear Arg linker into MM cells : F-MM cells a, those MM cells b. FurtheMM cells . AlthougMM cells c.Exosomes are internalized into cells via several pathways, including clathrin-mediated endocytosis , lipid rluc+ siRNA complexes delivery  on luminescence activity. Cells were treated with Ab-conjugated siRNAs for 24 h and luciferase assays were conducted. The luminescence intensity of anti-CD63 mAb-conjugated luc+ siRNA decreased to 55.9 ± 1.8% that of anti-CD63 mAb-conjugated control siRNA (p < 0.005) . Two siRNAs targeting MYC mRNA, s9129 and s9130, suppressed its transcription, while siRNA s9129 was more effective than the latter; the optimal duration for siRNA s9129 treatment was 96 h. Both siRNAs targeting CTNNB1 (#1 and #2) also suppressed mRNA transcription, with siRNA #2 being more effective; the optimal duration for siRNA #2 treatment was 48 h  a. Next, was 48 h . Treatmeectively b.Recently, exosomes have been used as a method for drug cargo delivery ,15,16; hTaken together, our data show that the developed exosome-capturing Ab-conjugated siRNA complexes are useful for treatment of MM cells. Importantly, as targets of siRNAs can be altered according to the optimal molecules in specific cancers, we believe that this technology will be valuable for individualized medicine. Our future work will involve investigating the pharmacokinetics and pharmacodynamics of Ab-conjugated siRNA complexes as well as assessing of their therapeutic effects in vivo. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic malignancy.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection elicits antibodies (Abs) that bind several viral proteins such as the spike entry protein and the abundant nucleocapsid (N) protein. We examined convalescent sera collected through 6 months (~24wks) post-SARS-CoV-2 infection in children to evaluate changes in neutralization potency and N-binding.Outpatient, hospitalized, and community recruited volunteers < 18 years with COVID-19 were enrolled in a longitudinal study at Seattle Children’s Hospital. Analysis includes symptomatic and asymptomatic children with laboratory-confirmed SARS-CoV-2 infection who provided blood samples at approximately 4wks  and 24 wks  after diagnosis. We measured neutralizing Ab using an in-house pseudoneutralization assay and anti-N binding Ab using the Abbott Architect assay.Of 32 children enrolled between April 2020 and January 2021, 27 had no underlying immunocompromised state and 25 of these 27 children had symptomatic disease. Ten of 27 had a > 2-fold decrease neutralization titers between 4 and 24wks (most were < 10-fold); 12 had < 2-fold change; and 5 had neutralization titers that increased > 2-fold over time . All but one of these 27 children had detectable neutralizing activity at 24wks. Anti-N Abs were assessed for 25 children at 4wks and 17 children at 24wks (data pending for 14 samples); all children with paired samples had a > 1.75-fold Abbott index reduction at 24wks, and 5 children had no detectable anti-N Abs by 24wks . An additional 5 children with symptomatic disease had complicating immunosuppression or multiple blood transfusions; 2 had decreasing neutralizing titers, 2 increased, and 1 had no change . Anti-N Abs were undetectable for one child by 24wks (data pending for 4 samples) . No participants received COVID-19 vaccine.Figure 1. Pseusoneutralization titers in children over time.Figure 2. Nucleocapsid-binding antibody titers in children over time.We show neutralizing Abs wane to a small degree over 24wks post-SARS-CoV-2 infection and remain detectable in most children. In contrast, anti-N Abs decreased, becoming undetectable in some children by 24wks. These findings add to understanding of the natural history of SARS-CoV-2 immunity in children.* This study was supported by CDC BAA75D301-20-R-67897Jesse Bloom, PhD, Flagship Labs 77 (Consultant)Moderna (Consultant) Janet A. Englund, MD, AstraZeneca GlaxoSmithKline (Research Grant or Support)Meissa Vaccines (Consultant)Pfizer (Research Grant or Support)Sanofi Pasteur (Consultant)Teva Pharmaceuticals (Consultant)


Coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused a still evolving global pandemic. Given the worldwide vaccination campaign, the understanding of the vaccine-induced versus COVID-19-induced immunity will contribute to adjusting vaccine dosing strategies and speeding-up vaccination efforts.n = 250); we also investigated humoral and cellular immune responses in vaccinated individuals (n = 21) of this cohort 5 months post-vaccination and assayed NAbs levels in COVID-19 hospitalized patients (n = 60) with moderate or severe disease, as well as in COVID-19 recovered patients (n = 34).Anti-spike-RBD IgGs and neutralizing antibodies (NAbs) titers were measured in BNT162b2 mRNA vaccinated participants (We found that one (boosting) dose of the BNT162b2 vaccine triggers robust immune  responses in COVID-19 convalescent healthy recipients, while naïve recipients require both priming and boosting shots to acquire high antibody titers. Severe COVID-19 triggers an earlier and more intense (versus moderate disease) immune response in hospitalized patients; in all cases, however, antibody titers remain at high levels in COVID-19 recovered patients. Although virus infection promotes an earlier and more intense, versus priming vaccination, immune response, boosting vaccination induces antibody titers significantly higher and likely more durable versus COVID-19. In support, high anti-spike-RBD IgGs/NAbs titers along with spike (vaccine encoded antigen) specific T cell clones were found in the serum and peripheral blood mononuclear cells, respectively, of vaccinated individuals 5 months post-vaccination.These findings support vaccination efficacy, also suggesting that vaccination likely offers more protection than natural infection.The online version contains supplementary material available at 10.1186/s12916-021-02090-6. Briefly, the kit measures responses to six different but overlapping peptides pools to cover protein sequences of six different SARS-CoV-2 antigens, without HLA restriction, and includes negative and positive controls; peptide sequences with high homology to endemic coronaviruses have been removed from the sequences, but sequences that may have homology to SARS CoV-1 were retained. Cells were incubated with antigens, and interferon-γ secreting T cells  were detected by measuring blue spots in each well by an independent operator. As per a manufacturer’s trial, PCR confirmed COVID-19 subjects showed high levels of reactivity with 23.2 % within 8–20 spots and a majority (58.9 %) with > 20 spots. Because the T-SPOT test uses fresh cells, we assayed the presence of SARS-CoV-2 S and N antigen-specific T cell clones 5 months post-vaccination; at this time point, we also measured anti-S-RBD IgGs and NAbs titers in donors’ serum.PBMCs from selected vaccine recipients . Results in figures are plotted as median values with 95% confidence interval. For statistical analysis, one-way ANOVA tests were performed unless otherwise stated. n = 250) anti-S-RBD IgGs and NAbs titers were more robust in females and showed a negative correlation with increasing age (not shown).Our screening for anti-S-RBD IgGs titer in the cohort of vaccinated health care workers  individuals reported a positive qRT-PCR test; all others were qRT-PCR negative. Healthcare workers included in this study were tested for SARS-CoV-2 qRT-PCR positivity periodically; in the case of COVID-19 related symptoms, all were tested by frequent qRT-PCR tests. The mean time since qRT-PCR testing for donors of the P1 group was 6 ± 4.51 months (range 1.5–11 months) and for all donors (P1/P2 groups) 4.19 ± 3.44 months (range 1.5–11 months). From those, only 7  were found also positive for anti-S-RBD-IgGs, whereas the rest were negative. On the other hand, 3 donors positive for anti-S-RBD IgGs at D1 did not report any SARS-CoV-2 related qRT-PCR test (asymptomatic/unsuspected virus carriers); similarly, 9 individuals who reported SARS-CoV-2 qRT-PCR positivity at D1 were found negative for NAbs  and in the merged P2/NEG (n = 240) groups at D22, D36, and D50  revealed that values higher than 8.315, 44.66, and 334.2 U/mL for anti-S-RBD IgGs predicted with significant sensitivity (> 90%) and specificity (> 96%) NAbs (%) inhibition values higher that 30%, > 50%, and 75% respectively ; (b) those with a disease of moderate severity, i.e., co-existing respiratory failure but moderate need for supplemental oxygen [up to minute ventilation (MV): 40%] ; and (c) those with severe disease marked by respiratory failure, requiring high flows of supplemental oxygenation , high-flow nasal oxygen (HFNO), and or intensive care unit (ICU) admission . In all cases, we observed a gradual increase in anti-S-RBD-IgGs and NAbs development which was however significantly more intense for both anti-S-RBD-IgGs and NAbs at D7 in patients with severe disease  further supporting the notion of sustained immunity post-SARS-CoV-2 infection.Given the significant positive correlation between anti-S-RBD IgGs and NAbs kinetics  versus those obtained after BNT162b2 vaccination in the P2/NEG group (see above) (n = 240). Given a reported median duration for symptoms initiation  in recruited (this study) COVID-19 patients upon hospitalization of ~ 9 days, we assumed that the measured NAbs titers correspond to D10 , D16 , and D39  post infection . Therefore, these time points roughly correspond to levels at D8, D22, and D36 post-vaccination of naïve BNT162b2 vaccinated donors. Accordingly, NAbs’ values from COVID-19 recovered patients’ isolated plasma (median of ~ 60 days post symptoms initiation) were compared to values obtained at D50 after the first dose of the BNT162b2 vaccine. As shown in Fig. P < 0.0001). In support, NAbs’ titers were significantly higher at D50 following vaccination versus plasma from COVID-19 recovered patients. The intensity of the secondary antigen-related immune responses was further evident by comparing  the kinetics of NAbs production in COVID-19 recovered patients receiving one dose of the vaccine versus naïve recipients receiving the second dose of the vaccine  after SARS-CoV-2 infection versus BNT162b2 vaccination, we used titers at D1-D30 for all COVID-19 patients  n = 20. Given ve) n = 20. Given ve) n = 20. Given ve) n = 20. Given ve) n = 20. Given n = 21) 5 months post-vaccination, anti-S-RBD IgGs/NAbs titers and assayed (in isolated PBMCs) the existence of SARS-CoV-2 S protein (vaccine delivered antigen) specific T cell clones. Our analyses revealed that all vaccine recipients were positive (> 0.8 U/mL)  for anti-S-RBD IgGs , it is evident that the presence of anti-RBD IgGs does not indicate anti-virus neutralizing activity. Thus, ideally, both assays should be employed to verify genuine immune protective responses against SARS-CoV-2 infection or following vaccination. The adaptation of this strategy is important to identify true COVID-19 convalescent recovered patients, while, regarding the qRT-PCR positive for SARS-CoV-2 infection individuals who showed no adaptive humoral immune responses (and thus remain COVID-19 naïve vaccine recipients), they surely require a prime-boost immunization strategy.+ and CD8+ T cells and antibody responses are protective, whereas uncoordinated responses frequently fail to control disease  at the indicated time points in vaccinated recipients (n=21) assayed at M5 for the presence of SARS-CoV-2 S protein specific T cells clones. Table S1. BNT162b2 mRNA vaccinated participants (n=250). Table S2. COVID-19 hospitalized patients (n=60).


Tumor metastasis is the most cause of high mortality for cancer patients. Identification of novel factors that modulate tumor cell migration is of great significance for therapeutic strategies. Here, we find that the ubiquitin-specific protease 8 (Usp8) promotes tumor cell migration through activating the c-Jun N-terminal kinase (JNK) pathway. Genetic epistasis analyses uncover Usp8 acts upstream of Tak1 to control the JNK pathway. Consistently, biochemical results reveal that Usp8 binds Tak1 to remove ubiquitin modification from Tak1, leading to its stabilization. In addition, human USP8 also triggers tumor cell migration and activates the JNK pathway. Finally, we show that knockdown of USP8 in human breast cancer cells suppresses cell migration. Taken together, our findings demonstrate that a conserved Usp8-Tak1-JNK axis promotes tumor cell migration, and providing USP8 as a potential therapeutic target for cancer treatment. Drosophila [scribbled (scrib) is silenced along the anterior/posterior compartment boundary in wing discs using ptc-gal4 driver. Knockdown of scrib confers wing disc cells invasive characteristics [Cancer has become the leading cause of human death worldwide . Tumor mosophila . To estaDrosophila, the ligand Eiger (Egr) binds its receptors Wengen (Wgn) or Grindelwald (Grnd) to trigger the downstream JNKKKs, including transforming growth factor-β-associated kinase 1 (Tak1), Slipper (Slpr), Wallenda (Wnd) and apoptotic signal-regulating kinase 1 (Ask1). In turn, JNKKKs activate the downstream JNKKs, including Hemipterous (Hep) and Mkk4, which sequentially phosphorylate and activate the Drosophila sole JNK, Basket (Bsk) [matrix metalloproteinase 1 (mmp1), which encodes a proteinase that hydrolyzes proteins in the extracellular matrix to trigger cell migration [As a member of the mitogen-activated protein kinase (MAPK) family, the evolutionarily conserved JNK pathway is involved in various cellular processes, such as apoptosis, migration, proliferation, and differentiation , 6. The et (Bsk) . Activatigration . Althougα-Synuclein [The process of ubiquitination requires three classes of enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases . Throughynuclein , and CHMynuclein . Furtherynuclein , 24. Albscrib-RNAi-caused tumor cell migration, while knockdown of usp8 exerted an opposite effect. Next, we uncovered that Usp8 was capable of activating the JNK pathway, and inhibition of the JNK pathway could suppress Usp8-enhanced tumor cell migration. Genetic analyses showed that Usp8 localized upstream of Tak1 to activate the JNK pathway. Further biochemical results revealed that Usp8 interacted with Tak1 to deubiquitinate it, culminating in Tak1 stabilization. Human USP8 also promoted scrib-RNAi-caused tumor cell migration and activated the JNK pathway in Drosophila wing discs, suggesting a conserved role of USP8 in regulating JNK signaling in mammalian system. Finally, we demonstrated that knockdown of USP8 in human breast cancer cells suppressed cell migration and downregulated the JNK pathway activity. Taken together, our study uncovers a conserved Usp8-Tak1-JNK axis to promote tumor cell migration, and provides USP8 as a potential therapeutic target for JNK-related cancers.Here, we found that overexpression of Usp8 promoted Drosophila deubiquitinases. Then, the transgene was ectopically expressed driven by patched-gal4 , which expresses along the anterior/posterior (A/P) compartment boundary in the wing disc. After screening, we found that overexpression of usp8 induced mild cell migration . Compared with the control , leading to activation of the JNK pathway [DN), in which the lysine on 53 is substituted by an arginine to suppress the endogenous JNK signaling [usp8 alone  phenotype in adult wings  experiments. The co-IP result revealed that Myc-tagged Tak1 (Myc-Tak1) was able to pull down Fg-tagged Usp8 (Fg-Usp8) Fig. . ReciproUsp8 protein contains a Usp8 dimerization domain (Usp8 dimer) domain in its N-terminus, a ubiquitin carboxyl-terminal hydrolase (UCH) domain in its C-terminus Fig. . To deteThe interaction between Usp8 and Tak1 prompted us to examine whether Tak1 is a substrate of Usp8. First, we used the translation inhibitor cycloheximide (CHX) to block protein synthesis and determine the stability of Tak1 with or without Usp8 co-expression. Strikingly, Tak1 protein was unstable, with a half-life of about 3 h Fig. . HoweverDrosophila to mammals [USP8 in the wing disc activated puc-lacZ expression  showed that the protein expression level of USP8 positively correlated with TAK1 in human breast cancer samples   and pJNK levels , without affecting total c-Jun levels . In line with our speculation, overexpression of ion Fig. . In addiDrosophila Usp8 to activate the JNK pathway and trigger cell migration. Finally, we uncovered that human USP8 positively regulates breast cancer cell migration through stabilizing TAK1. Taken together, our study reveals Usp8 is a novel regulator of the JNK pathway, provides human USP8 as a promising drug target for JNK-related breast cancer treatment.Herein, we find that Usp8 promotes tumor cell migration through activating the JNK pathway. Genetic and biochemical analyses show that Usp8 binds Tak1 to remove polyubiquitin chains from Tak1, culminating in Tak1 stabilization and the JNK pathway activation Fig. . Furtherusp8 in the wing fails to produce any obvious defects, possibly due to limited RNAi efficiency or functional redundancy. As a matter of fact, to overcome the shortcomings of RNAi-mediated screening, we generate transgenic flies to express Drosophila deubiquitinases for screening. Through this screening, we identify that Usp8 is an important modulator for JNK-mediated tumor cell migration. Given that the JNK pathway plays role in multiple physiological processes, it is also acceptable that Usp8 is involved in development. Loss of usp8 leads to embryonic lethality [usp8 during different developmental stages.Here, we find that Usp8 overexpression results in the loss of the anterior cross-vein (ACV) in adult wings, suggesting a physiological role of Usp8 in wing development. However, the knockdown of ethality , indicatα-Synuclein [Several substrates of Usp8 have been identified, including Smo , 19, Hrsynuclein , and CHMynuclein . Here, wynuclein , 33. ThePrevious studies have demonstrated Tak1 protein undergoes distinct linkages of polyubiquitination , 30. TheIncreasing studies have revealed that the elevated expression of human USP8 correlates with tumorigenesis, including proliferation and metastasis . USP8 prTaken together, our findings demonstrate that a conserved Usp8-Tak1 module promotes tumor cell migration, and provides USP8 as a potential therapeutic target for cancer treatment.To generate Fg-Usp8, Myc-Tak1, Myc-Bsk, Myc-Hep, Myc-Traf2 and HA-Ub constructs, the corresponding coding sequences were amplified by the DNA polymerase , and cloned them into the pcDNA3.1-Fg, pcDNA3.1-Myc or pcDNA3.1-HA backbone vectors, respectively. Truncated constructs including Usp8-N, Usp8-C, Tak1-N, Tak1-C were created by inserting the indicated coding sequences into the corresponding backbone vectors. To generate the Fg-USP8 and Myc-TAK1 constructs for mammalian cell expression, the corresponding full-length coding sequences were amplified from 293 T cell cDNA and cloned into pcDNA3.1-Fg or pcDNA3.1-Myc vectors, respectively.ptc-gal4, Ay-gal4, mirr-gal4, UAS-GFP had been described in our previous studies [-usp8 and usp8-RNAi  were kindly provided from Dr. Junzheng Zhang’s lab [scrib RNAi and traf2 RNAi flies were obtained from Dr. Lei Xue’s lab [puc-lacZ , UAS-bskDN , bsk RNAi , hep RNAi , tak1 RNAi  were purchased from Bloomington Drosophila Stock Center (BDSC), Kyoto Drosophila Stock Center (KDSC), Vienna Drosophila Stock Center (VDRC), Fly stocks of National Institute of Genetics (NIG), or Tsinghua Fly Center (THFC). The UAS-Fg-USP8 transgenic fly was generated by injection of UAS-attB-Fg-USP8 construct into the Drosophila embryos using phiC31 integration system. All the stocks were reared on standard Drosophila media at 25 °C. For all fly cross experiments, the indicated gal4 virgin flies were crossed with male flies of corresponding RNAi or transgenes at 25 °C.The  studies , 39. UASng’s lab . The scrue’s lab . puc-lacβ-Galactosidase , rat anti-Ci , mouse anti-Mmp1 , rabbit anti-pJNK  and DAPI . Mouse anti-Usp8 (1:50) was generated using aa1-333 of Usp8 protein as the antigen according to the method previously described [Immunostaining of imaginal discs were performed according to the method previously described . Wing anescribed . Mouse a2 conditions. The 293 T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS and 1% penicillin/streptomycin (PS). The MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM/F-12 (1:1) medium (Gibco) containing 10% FBS and 1% PS. Plasmids were transfected into the 293 T and breast cancer cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were collected 48 h after transfection and used for immunoprecipitation (IP) and immunoblotting (IB) analysis according to standard protocols. Antibodies used for IP and IB were as follows: mouse anti-Fg , mouse anti-Myc , mouse anti-HA , mouse anti-Actin , mouse anti-Usp8 , rabbit anti-USP8 , rabbit anti-TAK1 , rabbit anti-MMP9 , rabbit anti-pJNK , rabbit anti-c-Jun , rabbit anti-phospho-c-Jun (Ser63) , rabbit anti-phospho-c-Jun (Ser73) , rabbit anti-Lamin B , rabbit anti-β-tubulin , goat anti-mouse HRP , and goat anti-rabbit HRP . USP8 inhibitor  was purchased from MedChem Express .The 293 T cell line and five human breast cancer cell lines  were purchased from the American Type Culture Collection . All the cells were cultured in a humidified incubator at 37 °C with 5% COUSP8 in human breast cancer cells, two different siRNAs were used respectively. The siRNAs were synthesized by GenePharma , and the sequences were as follows: mock-siRNA: 5ʹ-UUC UCC GAA CGU GUC ACG UdTdT-3ʹ; USP8-siRNA#1: 5′-GGG UCA AUU CAA AUC UAC AdTdT-3′; USP8-siRNA#2: 5'-ACC GAA ACU GUU AUC AGG AUG AUA UdTdT-3' [To knock down UdTdT-3' . siRNAs Escherichia coli BL-21 cells; fusion proteins were induced expression by isopropyl β-D-thiogalactoside (IPTG) and purified using the Beaver beads GSH (Beaverbio) as previously described [The GST-vector and GST fusion protein plasmids were transfected into escribed . GST fusThe protein stability assay was conducted as previously described . In brie® Q RT SuperMix with gDNA wiper (Vazyme) following the manufacturer’s instructions. The quantitative real-time PCR (qRT-PCR) experiment was conducted with ChamQ SYBR® Color qPCR Master Mix (Vazyme) by the LightCycler® 96 System . The primers used for qRT-PCR were as follows: ACTIN, 5'-GAT CAT TGC TCC TCC TGA GC-3' (forward) and 5'-ACT CCT GCT TGC TGA TCC AC-3' (reverse), USP8, 5'-TTC CAT TCA ATA CTT GGA CCT GG-3' (forward) and 5'-CCA AAG AGC CTT TAG CCA ATG T-3' (reverse), TAK1, 5'-CCG GTG AGA TGA TCG AAG CC-3' (forward) and 5'-GCC GAA GCT CTA CAA TAA ACG C-3' (reverse), DUSP10, 5'-ATC GGC TAC GTC ATC AAC GTC-3' (forward) and 5'-TCA TCC GAG TGT GCT TCA TCA-3' (reverse), MMP9, 5'-AGA CCT GGG CAG ATT CCA AAC-3' (forward) and 5'-CGG CAA GTC TTC CGA GTA GT-3' (reverse). Relative quantification was calculated by the comparative 2−ΔΔCt method [Total RNA was isolated from breast cancer cells by TRIzol reagent (Invitrogen) according to the standard protocols. The cDNA template was synthesized using 1 µg of total RNA by HiScriptt method , and the5 cells) were planted into the upper chamber with serum-free medium after transfection for 48 h. Meanwhile, a medium with 10% FBS was added in the lower chamber. Then, the cells were cultured at 37 °C for 12 h (MDA-MB-231 cells) or 48 h (MDA-MB-468 cells). Migrated cells were fixed with 20% methanol for 20 min and stained with crystal violet (0.1%). The invasion assay was performed using the tissue culture plate inserts (TCS003024)  according to the manufacturer’s instructions. The image was acquired under a light microscope and the numbers of migrating cells were counted by the Image J software.The MDA-MB-231 or MDA-MB-468 cells .Quantification of the band intensity of immunoblotting was performed using the Image J software. Statistical analysis was conducted by the software of GraphPad Prism 7.0 . All the data shown in the figures were representative of at least three independent replicates and shown as the means ± SD. Unpaired student’s Supplementary materialsreproducibility-checklist


Poethko-Müller C, Buttmann-Schweiger N, Takla A (2018)Journal of Health Monitoring 3(4): 79-86.DOI 10.17886/RKI-GBE-2018-102An earlier version of this Fact sheet gave the following incorrect figures on page 81: ‘Whereas the vaccination coverage of 11- to 17-year-old girls in the Eastern German federal states is 55.6%, a considerably lower proportion of girls in Western Germany has received the complete HPV vaccination series (40.0%) (Table 1).’These figures incorrectly reported the coverage of ‘at least one single HPV vaccination dose’.The correct sentence reads: ‘Whereas the vaccination coverage of 11- to 17-year-old girls in the Eastern German federal states is 43.6%, a considerably lower proportion of girls in Western Germany has received the complete HPV vaccination series (29.5%) (Table 1).’DOI 10.17886/RKI-GBE-2018-102.2The corrected version of the article is available at


Nuclear translocation of AURKA is a prerequisite for RNA aberrant splicing, specifically triggering RBM4 splicing from the full isoform (RBM4-FL) to the short isoform (RBM4-S) in a kinase-independent manner. RBM4-S functions as a tumor promoter by abolishing RBM4-FL-mediated inhibition of the activity of the SRSF1-mTORC1 signaling pathway. Mechanistically, AURKA disrupts the binding of SRSF3 to YTHDC1, resulting in the inhibition of RBM4-FL production induced by the m6A-YTHDC1-SRSF3 complex. In turn, AURKA recruits hnRNP K to YTHDC1, leading to an m6A-YTHDC1-hnRNP K-dependent exon skipping to produce RBM4-S. Importantly, the small molecules that block AURKA nuclear translocation, reverse the oncogenic splicing of RBM4 and significantly suppress lung tumor progression. Together, our study unveils a previously unappreciated role of nuclear AURKA in m6A reader YTHDC1-dependent oncogenic RNA splicing switch, providing a novel therapeutic route to target nuclear oncogenic events.Aberrant RNA splicing produces alternative isoforms of genes to facilitate tumor progression, yet how this process is regulated by oncogenic signal remains largely unknown. Here, we unveil that non-canonical activation of nuclear AURKA promotes an oncogenic RNA splicing of tumor suppressor RBM4 directed by m AURKA interferes with the YTHDC1-SRSF3 complex, thus inhibiting the production of tumor-suppressing isoform RBM4-FL. In turn, AURKA links the interaction between hnRNP K and YTHDC1, thereby enhancing the production of tumor-promoting isoform RBM4-S. AURKA nuclear translocation inhibitors reverse the oncogenic splicing of RBM4 and significantly suppress lung tumor progression. Together, our findings reveal that the m6A reader YTHDC1-directed RBM4 aberrant splicing is triggered by nuclear AURKA, providing novel opportunities for targeted therapy of lung cancer by blocking nuclear oncogenic signaling.Aurora kinase A (AURKA) plays an important oncogenic role in promoting tumor initiation and progression.15 showed that RNA alternative splicing regulation ranked first  staining showed that AURKA was highly accumulated in the nuclei of cancer tissues compared to adjacent tissues Fig. . ConsistP-value < 0.05), including skipped exon (SE), alternative 3’ ss exon (A3E), alternative 5’ ss exon (A5E), mutually exclusive exons (MXE), and retained intron (RI) , which contains only an N-terminal RNA-binding domain compared to the full-length isoform (RBM4-FL)  Fig. . To deteFL) Fig. . ConsistFL) Fig. .We further investigated whether nuclear AURKA is required for RBM4 splicing regulation. To this end, we reconstituted AURKA-WT (wild-type), AURKA-NLS , and AURKA-NES (nuclear export sequence) in endogenous AURKA depleted NCI-H460 and A549 cells. We found that nuclear relocalized AURKA could effectively inhibit AURKA knockdown-mediated RBM4 exon inclusion, whereas cytoplasmic accumulation of AURKA could not affect it, suggesting that nuclear translocation of AURKA plays an essential role in regulating RBM4 splicing switch Fig. . In addiRBM4 gene has 95% homology with the human orthologue,20 we firstly explored whether RBM4-S isoform is widely expressed in mouse tissues. RT-PCR results showed that RBM4-S expresses in a tissue-specific manner   and YTHDC1-DM (m6A binding region mutant) in YTHDC1 depleted A549 cells, and found that re-expression of YTHDC1 wild-type, but not mutant, could rescue YTHDC1 knockdown-mediated RBM4 exon 3 skipping  region of RBM4 pre-mRNA , US drug collection (1280), Natural product screening library (120), Epigenetic library (43), Stemness library (303), and Autophagy library (150) in the EGFP-AURKA stable expression system to identify small molecules that could efficiently block AURKA nuclear translocation Fig. . IntriguSubsequently, we investigated whether JNJ and PHA could specifically modulate RBM4 splicing. We examined the expression level and kinase activity of AURKA in response to these two chemicals in A549 and NCI-H460 cells. Our results showed that JNJ and PHA inhibited AURKA kinase activity, but had no effect on the total expression level of AURKA  were subcutaneously injected into 4–6-weeks-old BALB/C (nu/nu) female nude mice, and the mice were randomly divided into four groups after the tumor volume reached more than 100 mm3. DMSO, VX680, VX680 + PHA, VX680 + JNJ were subsequently administered by gavage once a day for 10 days. Remarkably, tumor growth, as indicated by tumor volume and weight, was significantly suppressed with the combined drugs as compared to VX680 alone and the control group  in 8 pairs of clinical specimens  specimens and adjacent normal tissues to measure the protein levels of AURKA and RBM4 two splicing isoforms. Compared with adjacent normal tissues, the relative protein levels of AURKA and RBM4-S were evidently increased in tumor tissues, while the relative protein levels of RBM4-FL were evidently decreased in tumor tissues Fig. . The subens Fig. . We thenens Fig. . Hence, 6A reader YTHDC1-dependent mechanism in lung cancer cells, thereby providing a promising avenue for lung cancer therapeutics by targeting nuclear oncogenic signals. Nuclear translocation of AURKA acquires the kinase-independent function to promote RBM4 aberrant RNA splicing from the full isoform (RBM4-FL) to the short isoform (RBM4-S). RBM4-S enhances lung cancer cell proliferation by antagonizing RBM4-FL-meditated tumor suppression through eliminating the inhibition effect of RBM4-FL on the SRSF1-mTORC1 signaling pathway. Mechanistically, nuclear AURKA disrupts the binding of SRSF3 to YTHDC1 and bridges the interaction between hnRNP K and YTHDC1, resulting in the inhibition of m6A-YTHDC1-SRSF3-mediated RBM4 exon inclusion and the promotion of m6A-YTHDC1-hnRNP K-dependent RBM4 exon skipping. Furthermore, we show that AURKA nuclear translocation inhibitors could significantly suppress lung tumor progression by reversing the oncogenic splicing of RBM4.It is well-established that aberrant RNA splicing can produce abnormal proteins that contribute to cancer development. Till now, how this process is triggered by oncogenic signaling, however, has remained elusive. Here, we demonstrate a non-canonical function of nuclear AURKA to trigger an oncogenic RNA splicing of tumor suppressor RBM4 in a novel m14 Till now, a lot of efforts have been made to develop inhibitors against its kinase activity. However, the efficacy of AURKA kinase inhibitors such as Alisertib has been still very poor in clinical trials.33 The oncogenic function of AURKA is not completely blocked by the inhibition of its kinase activity, suggesting that AURKA may have non-classical kinase-independent carcinogenicity. Recent studies have shown that AURKA can function biologically in a kinase-independent manner. For example, AURKA promotes replication assembly during DNA replication initiation by kinase-independent mechanism and participates in the regulation of G1-S cell cycle.34 Defects in spindle assembly during mitosis induced by AURKA deletion can be rescued by the kinase-dead mutant.35 AURKA binds to N-MYC37 and C-MYC38 proteins in the MYC family, protecting them from proteasome pathway degradation. Our previous studies have shown that AURKA enhances breast cancer stem-like properties via its kinase-independent mechanism by (1) promoting the transcription of MYC and FOXM1 downstream target genes, (2) stabilizing DROSHA mRNA level,17 suggesting that the non-kinase-dependent function of AURKA is essential for the development of tumors. In this project, two observations suggest that AURKA may promote tumor progression by regulating RNA alternative splicing. One is that AURKA expression is highly associated with spliceosome activity by Pearson correlation analysis of whole-genome gene expression profile  with spliceosome activity score  represents one of the most prevalent internal RNA modifications in eukaryotes.39 m6A is recognized by its readers, such as YTH domain-containing proteins, to regulate mRNA metabolic processes, including RNA splicing.44 YTHDC1, as a critical nuclear m6A reader, localizes in YT-bodies adjacent to nuclear speckles. YTHDC1-deficient oocytes exhibit widespread splicing defects, which could be mostly rescued by supplementing with wild-type YTHDC1, but not m6A binding domain mutant.13 In our project, we find that RBM4 pre-mRNA can be highly methylated and bind to YTHDC1 protein  Fig. . We findner Fig. . In the ner Fig. . These r48 and our data also support that JNJ and PHA inhibit AURKA kinase activity , human normal bronchial epithelial cell line HBE, breast cancer cell line MDA-MB-231, liver cancer cell line HepG2 and human embryonic kidney HEK-293T cell line were obtained from the American Type Culture Collection (ATCC). These cell lines were authenticated at ATCC before purchase by standard short tandem repeat DNA-typing methodology. Each cell line was cultured in a standard medium as recommended by ATCC. All cells were incubated at 37 °C in a humidified incubator containing 5% CO49 Nocodazole, puromycin and MG132 were purchased from Selleck.Doxycycline was purchased from Clonetech. MLN8237, VX680, JNJ-26854165, and PHA-680632 were purchased from Selleck Chemicals. AKI603 was designed and synthesized by our laboratory.The natural product screening library and Autophagy library were purchased from Selleck Chemicals. International drug collection, US drug collection, Epigenetic library, and Stemness library were purchased from Topscience.Eight pairs of lung cancer specimens and adjacent normal tissues were obtained from patients undergoing surgery, following informed consent from patients and approved by the Institutional Ethics Review Board of the First Affiliated Hospital of Dalian Medical University (PJ-KS-KY-2021-94). In addition, we also obtained primary human lung tumor tissue microarrays (HLugS120CS01) from Outdo Biotech , containing 60 lung cancer and 60 corresponding adjacent normal samples.6/100 μl in PBS) of A549-Vector, RBM4-FL and RBM4-S cells. Tumor formation was monitored for 34 days.Female BALB/C-nu mice (6 weeks old) were subcutaneously inoculated with equal amounts (2 × 106/100 μl in PBS containing 50% Matrigel) of NCI-H460 cells. When tumor’s volume reached more than 100 mm3, the animals were randomized into four treatment groups and treated as follows: PBS, VX680 (20 mg/kg/d), PHA (40 mg/kg/d) combined with VX680 (20 mg/kg/d), JNJ (20 mg/kg/d) combined with VX680 (20 mg/kg/d). The agents used in this study were administered by gavage and the treatment continued for 10 days. The tumor volumes were determined using calipers to measure the longest (length) and shortest (diameter) every three days and were calculated according to the standard formula (length × diameter2 × 0.5).Female BALB/C-nu mice (6 weeks old) were subcutaneously inoculated with equal amounts , and carried out in accordance with established institutional guidelines and approved protocols.Total RNA was extracted from DOX-induced AURKA knockdown NCI-H460 cells by using Trizol reagent . cDNA library preparation for total RNA was performed with the Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin kit . The paired-end reads were generated by the Illumina Hi-Seq 2000 platform and mapped to the human genome. Changes in splicing isoforms were analyzed by MISO pipeline.50 To construct the RBM4 minigene reporter, we used PCR reactions to amplify a fragment containing exon 2, part of intron 2, exon 3, part of intron 3 and exon 4 of RBM4, and ligated this fragment to the pCDNA3.1(+) and pLVX-DsRed-Monomer-N1 vectors. RBM4 minigene reporters containing mutations were obtained from wild-type RBM4 minigene reporter by performing PCR reactions and Dpn I enzyme digestion. Full-length and truncated RBM4 fragments were cloned from human genome cDNA and ligated into pLVX-DsRed-Monomer-N1 vector (Clontech) in a frame with Flag tag. The pLVX-AURKA-HA plasmid, GST-fused AURKA full-length (1–403 aa) and truncations , pLVX-AURKA-EGFP plasmid, pLVX-AURKA-NLS plasmid and pLVX-AURKA-NES plasmid were kindly provided by Dr. Fei-Meng Zheng .15 Additional AURKA truncation 333–403 aa was cloned from AURKA full-length and ligated into GST vector. pb533a-flag-YTHDC1 and pCMV-myc-YTHDC1-DM plasmids were kindly provided by Professor Yun-Gui Yang .12 PCDH-Flag-YTHDC1-WT and PCDH-Flag-YTHDC1-DM plasmids were cloned from pb533a-flag-YTHDC1 and pCMV-myc-YTHDC1-DM, respectively. PZW2C-CGG1 and PGZ3-CGG1 were kindly provided by Professor Yang Wang .51 SRSF1 with HA tag was generated by PCR amplification and subcloned into PCDH-CMV-MCS-EF1-Puro vector using NheI/NotI restriction enzyme sites. SRSF3 with an N-terminal Flag epitope was generated by PCR amplification and subcloned into PCDH-CMV-MCS-EF1-Puro vector using NheI/NotI restriction enzyme sites. The shRNA targeting SRSF3 was inserted into pLKO.1-puro by AgeI and EcoRI restriction sites. The Fidelity of all vectors was confirmed by DNA sequencing. Expression plasmids were transfected into cells using lipofectamine 2000 or lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. All primers used in this study were listed in Supplementary Table The shRNA fragment targeting AURKA was inserted into inducible pLKO-Tet-On vector.All the RNA interference fragments were synthesized and purchased from GenePharma. The target sequences of AURKA, RBM4-FL, hnRNP A1, hnRNP L, hnRNP A2B1, hnRNP U, hnRNP K, METTL3, YTHDC1, SRSF3, hnRNP C, IGF2BP3, and control siRNAs were listed in Supplementary Table HEK-293T cells were used for packaging lentivirus with the 2nd generation packaging system plasmid psPAX2 (Addgene) and pMD2.G (Addgene). Lentiviruses were concentrated by ultracentrifugation, and viral titer determined by serial dilutions. For infection with the lentivirus, infected cells were selected with Puromycin (2 μg/ml).Total RNA was extracted by using TRIzol reagent . cDNA was generated by using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit  according to the manufacturer’s instructions. PCR amplification was performed in a 20 μl reaction system by using 2×EasyTaq PCR SuperMix . ACTB was used as the internal control. All primers used in RT-PCR were listed in Supplementary Table Quantitative PCR was performed with SYBR green  at Agilent Mx3005P real-time PCR system (Agilent Technologies). Expression analysis in different cell types was performed using specific primers for each gene. All primers used in RT-qPCR were listed in Supplementary Table Analysis of RNA immunoprecipitation (RIP), RNA fluorescent in situ hybridization (FISH), RNA pull-down, methylated RNA-Immunoprecipitation (MeRIP), immunofluorescence (IF) analysis, western blot analysis, co-immunoprecipitation (Co-IP) analysis, cytoplasmic/nuclear protein extraction, immunohistochemistry (IHC) assay and H-score analysis, colony formation assay, CCK8 proliferative assay, and statistical analysisSupplementary_MaterialsSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary figure 7Supplementary figure 8Supplementary figure 9Supplementary figure 10Supplementary figure 11Supplementary figure 12Supplementary Table 1 SILACSupplementary Table 2 RNAXXXseqSupplementary Table 3 UniProt KeywordsSupplementary Table 4 AURKA-RIP-seqSupplementary Table 5 spliceosome activity correlation analysisSupplementary Table 6 Plasmid primersSupplementary Table 7 siRNASupplementary Table 8 RT-PCR and qPCR primersSupplementary Table 9 PCR primers for in vitro transcription


Scientific Reports 10.1038/s41598-021-96307-6, published online 17 August 2021Correction to: The original version of this Article contained an error in the Results and discussion, under the subheading ‘Electrical transport investigation’,33, 3- and 2-dimensional Variable Range Hopping 34, and modified VRH by Efros–Shlivoski35, which takes into account Coulomb interactions (ES-VRH).”“The electronic transport in our samples has been tested by four common theoretical models: Fluctuation Induced Tunneling (FIT)now reads:33, 3- and 2-dimensional Variable Range Hopping 34, and modified VRH by Efros–Shklovskii35, which takes into account Coulomb interactions (ES-VRH).”“The electronic transport in our samples has been tested by four common theoretical models: Fluctuation Induced Tunneling (FIT)The original Article has been corrected.


DCAF1 mRNA in primary human osteoblasts. DCAF1 3′-untranslated region luciferase activity and its expression were significantly decreased after miR-3175 overexpression but were augmented with miR-3175 inhibition in human osteoblasts and hFOB1.19 osteoblastic cells. miR-3175 overexpression activated Nrf2 signaling, causing Nrf2 protein stabilization, antioxidant response (ARE) activity increase, and transcription activation of Nrf2-dependent genes in human osteoblasts and hFOB1.19 cells. Furthermore, dexamethasone-induced oxidative injury and apoptosis were largely attenuated by miR-3175 overexpression in human osteoblasts and hFOB1.19 cells. Importantly, shRNA-induced silencing or CRISPR/Cas9-mediated Nrf2 knockout abolished miR-3175 overexpression-induced osteoblast cytoprotection against dexamethasone. Conversely, DFAC1 knockout, by the CRISPR/Cas9 method, activated the Nrf2 cascade and inhibited dexamethasone-induced cytotoxicity in hFOB1.19 cells. Importantly, miR-3175 expression was decreased in necrotic femoral head tissues of dexamethasone-taking patients, where DCAF1 mRNA was upregulated. Together, silencing DCAF1 by miR-3175 activated Nrf2 signaling to inhibit dexamethasone-induced oxidative injury and apoptosis in human osteoblasts.Activation of nuclear-factor-E2-related factor 2 (Nrf2) signaling can protect human osteoblasts from dexamethasone-induced oxidative injury. DDB1 and CUL4 associated factor 1 (DCAF1) is a novel ubiquitin E3 ligase for Nrf2 protein degradation. We identified a novel DCAF1-targeting miRNA, miR-3175. RNA pull-down, Argonaute 2 RNA-immunoprecipitation, and RNA fluorescent in situ hybridization results confirmed a direct binding between miR-3175 and  Dexamethasone (DEX) is commonly utilized in patients suffering from inflammatory and autoimmune diseases . Yet, loheme oxygenase 1 (HO1), NAD(P)H quinone oxidoreductase-1 (NQO1), glutathione (GSH), γ-glutamyl cysteine ligase catalytic subunit (GCLC), and modified subunit (GCLM), are antioxidant genes and detoxifying enzymes [In the unstimulated condition, nuclear-factor-E2-related factor 2 (Nrf2) is mainly inactivated in the cytoplasm, directly interacting with its suppressor protein Kelch-like ECH-associated protein 1 (Keap1). The latter mediates ubiquitination and proteasomal degradation of the Nrf2 protein –12. With enzymes –12.2O2) [Activation of Nrf2 cascade, using pharmacological agents, could ameliorate DEX-induced oxidative injury and death in osteoblasts/osteoblastic cells. Li et al. reported that SC79 activated Akt-dependent Nrf2 cascade to inhibit DEX-induced death of murine osteoblasts and MC3T3-E1 osteoblastic cells . Liu et 2O2) .MicroRNAs (miRNAs) are small (~22 nucleotides long) and conserved noncoding RNAs (ncRNAs), regulating gene expression by directly binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs , 19. TheDEX was obtained from Sigma-Aldrich . All cell culture reagents, including fetal bovine serum (FBS), DMEM, and antibiotics, were provided by Gibco Co. . Antibodies for HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), α-Tubulin (#2125), and Lamin B1 (#13435), the cleaved-poly (ADP-ribose) polymerase  and the cleaved-caspase-3 (#9664) were provided by Cell Signaling Tech . The anti-GCLC antibody (ab55435) was purchased from Abcam . From Shanghai Genechem Co. , the viral constructs, primers, and plasmids were obtained.The primary human osteoblasts were provided by Dr. Ji’s group at Nanjing Medical University , 21. The-ΔΔCt method was utilized for data quantification. GAPDH was always examined as the internal control and the reference gene. For detection of miR-3175 expression, a PrimeScript miRNA qRT-PCR Kit  was utilized, with U6 RNA examined as the internal control. The mRNA primers for the Keap1-Nrf2 cascade genes were provided by Dr. Liu at Jiangsu University [TRIzol reagents were added to extract total RNA, which was reverse-transcribed to cDNA. qRT-PCR assays were carried out through an SYBR Green PCR kit  under the ABI-7900HT Real-Time PCR System (Applied Biosystems). A 2iversity . Other vThe miR-3175 precursor sequence  or the anti-sense sequence (“antagomR-3175”) was inserted into a GV-369 lentiviral construct (Genechem). The construct, along with the lentivirus Helper plasmids (psPAX2 and pMD2.G), were co-transfected to HEK-293T cells. Virus in culture supernatants were enriched and filtered and was added to the primary human osteoblasts or hFOB1.19 osteoblastic cells (cultured in polybrene-containing medium). Puromycin was added to selected stable cells. Expression of mature miR-3175 in stable cells was verified by qRT-PCR assays. The lentiviral microRNA control construct (“Vec”) or the microRNA anti-sense control (“antaC”) were utilized as controls.DCAF1 mRNA was tested by qRT-PCR assay, with its level normalized to the “Input” control.A Pierce Magnetic RNA pull-down Kit , 26 was DCAF1 mRNA probe were cotransduced to primary human osteoblasts (at 37 °C for 48 h). Cells were rinsed and observed under a fluorescence microscope .Fluorescent in situ hybridization (FISH) kit  was employed for RNA-FISH experiments. Briefly, the FITC (green fluorescence)-labeled miR-3175 probe and the Cy3 (red fluorescence)-labeled DCAF1 mRNA), and their levels normalized to the “Input” control.Lysates from the primary human osteoblasts (800 μg lysates per treatment) were precleared and incubated with magnetic beads conjugated with the anti-Argonaute 2 antibody . After 12 h, beads were washed and incubated with Proteinase K. Thereafter, qRT-PCR was performed to test the purified RNAs . Cells DCAF1 3′-UTR luciferase reporter activity in human osteoblasts with applied genetic modifications was tested by a Promega kit [A pGL4.13 (luc2/SV40) construct encoding DCAF1 3′-UTR was synthesized by Genechem . The construct was transfected to primary human osteoblasts or hFOB1.19 osteoblastic cells by Lipofectamine 3000  together with the Renillaluciferase reporter vector and pRL-SV40 . DCAF1 mega kit .The GV-369 lentiviral construct encoding the 3′-UTR-null DCAF1 was provided by Genechem  and was sequence-verified. The primary human osteoblasts were seeded into six-well plates at 60% confluence and were transduced with construct via Lipofectamine 3000. Stable cells were selected by puromycin. The expression of DCAF1 was verified by a Western blotting assay.Briefly, cell lysates were extracted, quantified (30 μg per treatment in each lane), and electro-transferred to 10% SDS-PAGE gels. The lysate proteins were then transferred to PVDF blots. After blocking, the blots were incubated with designated primary and corresponding secondary antibodies. To visualize signaling of antigen-antibody binding, the enhanced chemiluminescence (ECL) reagents (Sigma) were applied. An ImageJ software (downloaded from the NIH website) was used for data quantification.With mitochondrial depolarization JC-1, fluorescence dye will aggregate into mitochondria to form green monomers . FollowiHuman osteoblasts or hFOB1.19 osteoblastic cells were seeded into 96-well plates (at 4500 cells per well). Following treatment, a ssDNA ELISA kit  was utilized to quantify ssDNA contents. The ssDNA ELISA absorbance was tested at 450 nm in each well.Human osteoblasts or hFOB1.19 osteoblastic cells were seeded into six-well plates . Following treatment, cells were stained with 10 μM of CellROX . After 30 min, cells were washed and CellROX red fluorescence intensity was tested under a Fluoroskan Ascent fluorescence spectrofluorometer. CellROX fluorescence images were presented as well.The protocols of other cell functional assays, including CCK-8 assaying of cell viability, caspase-3 activity assay, and thiobarbituric acid reactants (TBAR) activity assaying of lipid peroxidation, as well as nuclear TUNEL staining and Annexin V FACS, were described in detail in our previous studies and elsewhere , 30, 31.To hFOB1.19 osteoblastic cells, the Nrf2 shRNA lentiviral particles  were added. Stable cells were then selected by puromycin. Nrf2 silencing was verified by qRT-PCR assays.hFOB1.19 cells were seeded into six-well plates at 60% confluence and were transduced with a CRISPR/Cas9-DCAF1-KO-GFP construct  or a CRISPR/Cas9-Nrf2-KO-GFP construct (from Dr. Tan ). GFP-pon = 20) written-informed consent DEX-treated patients. All patients underwent femoral head resection. miR-3175 and DCAF1 mRNA expression in fresh tissues was examined by qRT-PCR assays. Protocols were in according to the principles of the Declaration of Helsinki, with approval from the Ethics Committee of Hunan Provincial People’s Hospital.As described in ref. , human nt-test  was utilized to compare the significance between two treatment groups. P < 0.05 was considered statistically significant.Data were presented as mean ± standard deviation (SD). For comparison of multiple groups, statistical differences were analyzed by one-way ANOVA (multiple comparisons) with post hoc Bonferroni test (SPSS version 21.0). The unpaired DCAF1. Other miRNA databases, including miRBase, miRNAmap, and miRTarbase, were searched as well to verify the retrieved miRNAs. Three candidate DCAF1-targeting miRNAs were retrieved, miR-876-3p, miR-3175, and miR-1236-3p. The context score percentage was >98% and the context++ score was < −0.4 [DCAF1 silencing was examined. Bioinformatics studies and preexperimental results identified that miR-3175 potentially targets DCAF1’s 3′-UTR (at position 828–835)  Fig. . The bincan V7.2 , Fig. 1Acan V7.2 .Fig. 1miDCAF1 mRNA and miR-3175 both co-immunoprecipitated with the anti-Ago2 antibody in human osteoblasts. The nonspecific anti-IgG antibody did not associate DCAF1 mRNA and miR-3175  mainly in the cytosol of human osteoblasts  is a primary component of RNA-induced silencing complex (RISC) that facilitates miRNA binding to its target mRNA. It also promotes the cleavage of the target mRNAs by its endonuclease activity , 33. Ago175 Fig. . The RNAsts Fig. . Moreovests Fig. . Thus, RDCAF1 expression, a lentiviral construct encoding the miR-3175 precursor was transduced to human osteoblasts. Via selection by puromycin, two stable lines, OE-miR-3175-L1 and OE-miR-3175-L2, were established. qRT-PCR assays results, Fig. DCAF1 3′-UTR luciferase activity in human osteoblasts. Furthermore, DCAF1 mRNA levels were significantly decreased  potently decreased mature miR-3175 expression Fig. . ConversRNA Fig.  and protRNA Fig.  expressiRNA Fig.  robustlyity Fig.  and sileRNA Fig.  and protRNA Fig. . CollectNrf2 mRNA expression was unchanged  was transduced. The construct restored DCAF1 protein expression in miR-3175-overexpressed osteoblasts Fig. . ConsequNrf2 mRNA . Moreover, the CRISPR/Cas9 gene-editing method was employed to complete knockout Nrf2 in hFOB1.19 cells, and single stable cells were established (“koNrf2 cells”). As shown, miR-3175 overexpression-induced ARE activity increase Fig.  as well RNA Fig.  and protRNA Fig.  expressiRNA Fig. . UnsurprRNA Fig. . DEX-indRNA Fig.  was signRNA Fig. . These rDCAF1 KO screening, single stable cells were established: ko-DCAF1 cells. As shown, DCAF1 protein was depleted in ko-DCAF1 hFOB1.19 cells , HO1 (Pearson Correction = −0.79), and NQO1 (Pearson Correction = 0.69) mRNA downregulation (Nrf2 cascade inhibition).At last, expression of miR-3175 in necrotic femoral head tissues of DEX-taking human patients was tested by qRT-PCR assays. As shown miR-3175 expression in the necrotic femoral head tissues (“N”) was dramatically lower than that in matched surrounding normal bone tissues (“S”) Fig. . ConversHO1 Fig. , and NQOQO1 Fig.  mRNA levups Fig. . TherefoThe function and the potential targets of miR-3175 are still largely unknown. Qi et al. reported that miR-3175 acted as a tumor-suppressive miRNA and was downregulated in human glioma. Transfection of miR-3175 mimics induced proliferation inhibition and apoptosis in glioma cells possibly by inhibiting the PI3K-Akt pathway . Xia et DCAF1-targeting miRNA in human osteoblasts. RNA pull-down, Ago2 RNA-IP, and RNA-FISH experiment results all supported a direct binding between miR-3175 and DCAF1 mRNA in primary human osteoblasts. In human osteoblasts and hFOB1.19 osteoblastic cells, forced overexpression of miR-3175 inhibited DCAF1 3′-UTR luciferase activity and downregulated DCAF1 expression. Conversely, miR-3175 silencing by antagomiR-3175 increased DCAF1 expression. Importantly, the mutant miR-3175 mimics, containing mutations at the proposed binding sites to DCAF1 3′-UTR, failed to alter DCAF1 expression in human osteoblasts.The results of the present study indicated that miR-3175 is a novel HO1, NQO1, and GCLC). Yet transfection of the two mutant miR-3175 mimics failed to activate Nrf2 signaling in human osteoblasts. Moreover, restoring DCAF1 expression, by the UTR-null-DCAF1 construct, abolished miR-3175 overexpression-induced Nrf2 cascade activation in human osteoblasts. These results supported that DCAF1 silencing is the key mechanism responsible for Nrf2 cascade activation by miR-3175 (Fig. We further showed that silencing of DCAF1 by miR-3175 activated Nrf2 signaling in human osteoblasts and hFOB1.19 cells, causing Nrf2 protein stabilization, ARE activity increase, transcriptional activation of Nrf2-dependent genes (175 Fig. .Different miRNAs could protect osteoblasts/osteoblastic cells from DEX-induced oxidative injury and cell death. For example, Zhao et al. reported that Keap1 silencing by miR-200a activated Nrf2 signaling to protect osteoblastic cells from DEX . InhibitWe found that activation of Nrf2 signaling by miR-3175 potently inhibited DEX-induced oxidative injury in human osteoblasts. DEX-induced ROS production, lipid peroxidation, mitochondrial depolarization, and DNA damage were all largely attenuated in miR-3175-overexpressed human osteoblasts. Furthermore, forced overexpression of miR-3175 largely inhibited DEX-induced cytotoxicity and apoptosis in human osteoblasts. Importantly, we found that activation of the Nrf2 cascade was required for miR-3175-induced osteoblast cytoprotection against DEX. In hFOB1.19 cells, Nrf2 shRNA or KO abolished miR-3175 overexpression-induced osteoblast cytoprotection. Conversely, DFAC1 KO activated the Nrf2 cascade and inhibited DEX-induced cytotoxicity in hFOB1.19 cells. Significantly, miR-3175 overexpression failed to further increase Nrf2 cascade activation nor inhibiting DEX-induced cytotoxicity in DCAF1 KO hFOB1.19 cells. These results clearly demonstrated that DCAF1 silencing by miR-3175 activated Nrf2 signaling to inhibit DEX-induced oxidative injury and death in human osteoblasts Fig. .miR-3175 is a novel DCAF1-targeting miRNA. Silencing DCAF1 by miR-3175 activated Nrf2 signaling to inhibit DEX-induced oxidative injury and apoptosis in human osteoblasts.


The hypoxic microenvironment is presumed to be a sanctuary for myeloid leukemia cells that causes relapse following chemotherapy, but the underlying mechanism remains elusive. Using a zebrafish xenograft model, we observed that the hypoxic hematopoietic tissue preserved most of the chemoresistant leukemic cells following the doxorubicin (Dox) treatment. And hypoxia upregulated TFEB, a master regulator of lysosomal biogenesis, and increased lysosomes in leukemic cells. Specimens from relapsed myeloid leukemia patients also harbored excessive lysosomes, which trapped Dox and prevented drug nuclear influx leading to leukemia chemoresistance. Pharmaceutical inhibition of lysosomes enhanced Dox-induced cytotoxicity against leukemic cells under hypoxia circumstance. To overcome lysosome associated chemoresistance, we developed a pH-sensitive dextran-doxorubicin nanomedicine (Dex-Dox) that efficiently released Dox from lysosomes and increased drug nuclear influx. More importantly, Dex-Dox treatment significantly improved the chemotherapy outcome in the zebrafish xenografts transplanted with cultured leukemic cells or relapsed patient specimens. Overall, we developed a novel lysosome targeting nanomedicine that is promising to overcome the myeloid leukemia chemoresistance.The online version contains supplementary material available at 10.1186/s13045-021-01199-8. The mechanism(s) of how hypoxia regulates chemoresistance remains unclear, and the potential targeting therapeutic strategy is poorly developed , 2. The The two days post-fertilization (2dpf) zebrafish embryos are immunodeficient due to the absence of adaptive immune system  and werehif1al were highly enriched in CHT  and the hypoxia-associated genes CHT Fig. , indicatWe next explored the role of lysosome in regulating leukemia chemoresistance. Lysosome inhibitor bafilomycin (Baf) or chloroquine (CQ) efficiently decreased the ratio of LysoTracker- or LysoSensor-high K562 cells  of xenografted zebrafish. (A–F) Kasumi-1 or OA3 cells were microinjected into 2dpf embryos and the fluorescent intensities of leukemic cells localized in CHT (highlighted by the yellow box) and non-CHT at different time points post-injection were quantified. Some cells entered the yolk sac during microinjection and were excluded for counting. (G–J) Kasumi-1 xenografted zebrafish were treated with Dox at one day post-injection (1dpi), and after two days the leukemic cells in CHT were quantified for the fluorescent intensity (H), the cell number (I) and the mRNA expression of human ribosome gene L32 (J). (K–L) The DiI-labeled Kasumi-1 cells were xenografted into zebrafish embryos and treated with Dox before staining with TUNEL to identify the apoptotic cells. (K) The TUNEL+DiI+ cells in CHT and non-CHT were quantified and the ratio was calculated by dividing the total DiI+ cell number.Additional file 2. Figure S2. Hypoxic microenvironment characterized leukemic cells with enriched lysosomes. (A) The expressions of hypoxia-associated genes hif1al, hif1ab in 2dpf embryos and hematopoietic specific marker cmyb were detected by in situ hybridization. The CHT region was highlighted with yellow box. (B) The hypoxia-cultured K562 cells were examined by Western blot for expressions of lysosome-related genes LC3B, TFEB, LAMP1. (C–E) The hypoxia-cultured K562 cells were examined for the ratio of LysoTracker-high cells (C–D) and mRNA levels of lysosome-associated genes V-ATPase, LAMP1, LAMP2 (E). (F–H) The ratio of LysoTracker-high cells (F–G) and the lysosome gene expression (H) were significantly increased in hypoxic Kasumi-1 cells compared with normal condition.Additional file 3. Figure S3. Excessive lysosome prevents the Dox cytotoxicity. (A–B) K562 cells were treated with V-ATPase inhibitor bafilomycin (Baf) or lysosome inhibitor chloroquine (CQ). The ratio of LysoTracker high cells was quantified by flow cytometry. (C–D) K562 cells were treated Baf or CQ, and the ratio of LysoSensor high cells was quantified by flow cytometry. (E) The hypoxia-treated K562 cells have higher viability following Dox treatment, but the viability was significantly reduced in CQ+Dox or Baf+Dox. (F) K562 cells were labeled with DiI and xenografted into zebrafish embryos before treating with CQ, Dox or both. The K562 cells in CHT were quantified by fluorescent intensity at 48h post treatment.Additional file 4. Figure S4. Characterization of Dex-Dox/Dex-b-DOX nanoparticles. (A-B) We synthesized the oxidized dextran (Dex-CHO) with dextran of different molecular weights (Dex5k/20K/70K/150K) and then conjugated with Dox. As a negative control, we reduced the pH-sensitive imine bond (-C=N-) in Dex-Dox to obtain the pH-insensitive carbon-nitrogen bond (-C-N-) in Dex-b-Dox. (A) The peak at 9.6 ppm in 1H NMR results proved the successful synthesis of Dex-CHO. In Dex-Dox or Dex-b-Dox the benzene peaks of Dox appeared at around 7.8 ppm while the Dex-CHO peak at 9.6 ppm disappeared, which proved their successful synthesis. (B) The representative peaks of Dex-Dox, Dex-b-Dox or Dex-CHO were at 1640 cm-1(v-C=N-), 1210 cm-1(v-C-N-) or 1720 cm-1(v-C=O-) in the Fourier transform infrared (FT-IR) spectrum. (C) In dynamic light scattering (DLS) results the particle size of Dex5k/150k-Dox or Dex5k/150k-b-Dox was respectively 35 nm or 60 nm, both with the small polymer dispersity index (PDI). (D–E) Dex5k, Dex40k, Dex70k, and Dex150k have similar CHO contents, quantified by oxidation degree, and Dox drug loading (E). But Dex20k-Dox and Dex70k-Dox have relatively larger particle sizes and PDI than Dex5k-Dox and Dex150k-Dox in DLS assay (D), resulting in precipitation and poor stability. Therefore, we cho se Dex5k-Dox and Dex150k-Dox for the follow-up experiments. (F) Typical transmitted electron microscopy (TEM) images of Dex5k/150k-Dox and Dex5k/150k-b-Dox. (G) Time and pH-dependent  Dox release profiles of Dex5k/150k-Dox and Dex5k/150k-b-Dox in vitro (n = 3).Additional file 5. Figure S5. The pH-responsive Dex-Dox nanomedicine efficiently eliminate hypoxic leukemic cells in vitro. The levels of cell viability , apoptosis  and ROS labeled by oxidative detective reagent DCFH  were measured in Dox or Dex5k/150k treated K562 or Kasumi-1 cells (n = 3). (G) The hypoxia-cultured Kasumi-1 cells have higher viability post Dox treatment, but the viability was significantly reduced in Dex5k/150k-Dox (n = 3). (H) The Kasumi-1-xenografted-zebrafish embryos were treated with Dox or Dex5k/150k-Dox from 1dpi to 3dpi, and leukemic cells in CHT were counted for fluorescence intensity. (I) The K562-xenografted-zebrafish embryos were pretreated with CQ before adding Dex5k-Dox from 1dpi to 3dpi, and leukemic cells in CHT were quantified for fluorescence intensity.Additional file 6. Figure S6. Dox and Dex-Dox similarly impaired lysosome acidification of leukemia cells. (A–B) The K562-xenografted zebrafish were treated with Dox or Dex Dex5k-Dox before staining for TUNEL. The TUNEL+DiI+ cells in CHT and non-CHT were quantified and the ratio was calculated by dividing the total DiI+ leukemia cell number. (C–D) Hypoxia-cultured Kasumi-1 cells were treated with Dox or Dex5k/150k-Dox, stained with LysoSensor DND-189, followed by imaging with microscope (C) or analyzing by flow cytometry (D). Kasumi-1 cells in normoxia or hypoxia were gated in flow results to quantify the ratio of LysoSensor-high cells (E–F). (G–H) The normoxia-cultured or hypoxia-cultured K562 cells were gated in flow results to quantify the ratio of LysoSensor-high cells.Additional file 7. Figure S7. The pH-responsive Dex-Dox efficiently accumulated to eliminate chemoresistant leukemic cells in vivo. (A–B) The 2dpf zebrafish embryos were treated with DMSO, Dox or Dex5k-Dox for 24 h before imaging under microscope and the autonomous red fluorescence of Dox were quantified. (C–D) The GFP expressing Kasumi-1 cells were xenografted into zebrafish embryos and the Dox localization was imaged by red fluorescence. More Dex5k-Dox treated cells have red fluorescence compared with the Dox alone, suggesting Dex5k-Dox more efficiently delivered Dox into the CHT localized leukemia cells. (E–F) The zebrafish embryos were xenografted with the leukemic cells from two relapsed patients and treated with Dox or Dex5k/150k-Dox. The fluorescent intensity of leukemic cells in CHT was counted at two-day post-treatment.Additional file 8. Figure S8. Graphic abstract. By visualizing in a zebrafish xenograft model, we found that the chemoresistant leukemic cells were mainly accumulated in the hypoxic hematopoietic tissue (A). The hypoxic microenvironment characterized leukemic cells with excessive lysosomes, thereby sequestering the chemotherapeutics inside to reduce its cytotoxicity (left panel in C). We developed the pH-sensitive Dex-Dox nanomedicine to release Dox from lysosomes and enter the nucleus, thereby efficiently eliminating the chemoresistant leukemic cells in vivo .