Accumulating evidence showed that regulating tumor microenvironment plays a vital role in improving antitumor efficiency. Programmed Death Ligand 1 (PD-L1) is expressed in many cancer cell types, while its binding partner Programmed Death 1 (PD1) is expressed in activated T cells and antigen-presenting cells. Whereas, its dysregulation in the microenvironment is poorly understood. In the present study, we confirmed that evodiamine downregulates MUC1-C, resulting in modulating PD-L1 expression in non-small cell lung cancer (NSCLC).xenograft nude mice were used to assess the effect of evodiamine on tumorigenesis in vivo. Lewis lung carcinoma model was used to investigate the therapeutic effects of combination evodiamine and anti-PD-1 treatment.Cell viability was measured by MTT assays. Apoptosis, cell cycle and surface PD-L1 expression on NSCLC cells were analyzed by flow cytometry. The expression of MUC1-C and PD-L1 mRNA was measured by real time RT-PCR methods. Protein expression was examined in evodiamine-treated NSCLC cells using immunoblotting or immunofluorescence assays. The effects of evodiamine treatment on NSCLC sensitivity towards T cells were investigated using human peripheral blood mononuclear cells and Jurkat, apoptosis and IL-2 secretion assays. Female H1975 + T cell effector function. Meanwhile, evodiamine showed good anti-tumor activity in H1975 tumor xenograft, which reduced tumor size. Evodiamine exhibited anti-tumor activity by elevation of CD8+ T cells in vivo in Lewis lung carcinoma model. Combination evodiamine and anti-PD-1 mAb treatment enhanced tumor growth control and survival of mice.We showed that evodiamine significantly inhibited growth, induced apoptosis and cell cycle arrest at G2 phase of NSCLC cells. Evodiamine suppressed IFN-γ-induced PD-L1 expression in H1975 and H1650. MUC1-C mRNA and protein expression were decreased by evodiamine in NSCLC cells as well. Evodiamine could downregulate the PD-L1 expression and diminish the apoptosis of T cells. It inhibited MUC1-C expression and potentiated CD8+ T cells and downregulating of the MUC1-C/PD-L1 axis. Our findings uncover a novel mechanism of action of evodiamine and indicate that evodiamine represents a potential targeted agent suitable to be combined with immunotherapeutic approaches to treat NSCLC cancer patients. MUC1-C overexpression is common in female, non-smoker, patients with advanced-stage adenocarcinoma.Evodiamine can suppress NSCLC by elevating of CD8Supplementary information accompanies this paper at 10.1186/s13046-020-01741-5. Despite progress and development of new treatment and drugs, only 17.7% of all patients with lung cancer are alive ≥5 years after diagnosis [Lung cancer in the world is the most commonly diagnosed carcinoma and becomes a paramount cause of cancer death, which is frequently associated with poor clinical outcomes –3.. Despiagnosis –6. Blockiagnosis . In addiiagnosis –11. Actiiagnosis , 13. Theiagnosis . Howeveriagnosis , 16. Theiagnosis . Increasiagnosis –22.Increasing evidence has indicated that MUC1-C plays a critical role in anti-cancer properties; for example, the transcriptional regulation of genes associated with tumor invasion , prolifeTetradium [Evodiamine is a novel alkaloid, which was isolated from the fruit of etradium , and it etradium . Severaletradium –36. HoweIn this study, the effects of evodiamine on cell viability, cell cycle, and apoptosis in the human NSCLC cell lines were investigated and the underlying mechanisms are further explored. More importantly, the anticancer and immunomodulatory activities of evodiamine in the human NSCLC cells in vitro and in vivo models are carefully examined. Our results confirm the efficacy of combining evodiamine and anti-PD-1 mAb treatment against NSCLC cells. Our research also explored the involvement of MUC1-C/PD-L1 signaling of evodiamine in anti-lung cancer. Evodiamine can improve immunity in vivo by inhibiting PD-L1 expression in cancer. Therefore, our findings disclose the inhibition of evodiamine anti-NSCLC, which might have potential clinical implications.Evodiamine was supplied by Selleck Chemicals  and was dissolved in dimethyl sulfoxide (DMSO) and stored at − 20 °C. Primary antibodies against GAPDH (#5174), PD-L1(#13684), MUC1-C(#16564), C-MYC(#18583) were provided by Cell Signaling Technology . Fluorescein secondary antibodies were provided by LI-COR Biosciences . Dead Cell Apoptosis Kit with Annexin V-FITC/PI were provided by BD Biosciences .The proliferation of lung cancer cells was assessed using the MTT assay as described previously . After tApoptosis was analysis as described previously . After tThe detailed procedure was reported previously . The fol4 cells. PBMCS were isolated from healthy donors by using Ficoll-Paque density centrifugation. Then, the obtained peripheral blood lymphocytes were added to the co-culture system at a ratio of 2:1. PBMC/lung cancer cell H1975 /H1650 co-cultured cells in six well plates were treated with evodiamine or vehicle. PBMC/H1975 (CshRNA), H1975 (MUC1-CshRNA) co-cultured cells and PBMC/H1650 (CshRNA), H1650(MUC1-CshRNA) co-cultured cells in 6 well plates were treated with evodiamine or vehicle. Cells were treated with evodiamine for 48 or 72 h. Afterward, lymphocyte cells were harvested from the co-culture system, and the T cells were stained for apoptosis assay.Human peripheral blood mononuclear cells (PBMC)/H1975 and H1650 cells were seeded at a density of 3 × 10Cells were transfected by using lentiviral vectors with control shRNAand MUC1-CshRNA. Puromycin was used for optimal selection of the transfected cells. For flow cytometry for cell cycle and apoptosis analysis, cells were stained with antibody in stain buffer for 30 min (in the dark at 4 °C). Cells were analyzed by BD FACSAria III flow cytometer (BD Biosciences). The surface expression of PD-L1 and MUC1-C were detected with flow cytometry.H1975 were treated with evodiamine for 24 h, and protein was extracted from cells by using NE-PER™ Nuclear and cytoplasm extraction reagents . The expression of PD-L1 and MUC1-C on H1975 was detected using Western blotting.After Total RNA isolated, cDNAs were synthesized by a cDNA Reverse Transcription SuperMix Kit (Bio-RAD) Using the Power SYBR Green PCR Master Mix (Roche) to detect the PD-L1 and MUC1-C mRNA expression. Primers used for qPCR were reported previously .H1975 and H1650 cells were transfected with PD-L1 promoter-Luciferase reporter expression (pD-L1-Luc) or mock vehicle (Active Motif). After treated with evodiamine for 48 h, the cells were lysed and analyzed by using the Dual-Luciferase® Reporter Assay System .H1975 and H1975 MUC1-C knockout were treated with IFN-γ for 24 h. Jurkat cells were activated and added to H1975 and H1650 cells at a ratio of 2:1 or 4:1 Jurkat: H1975/H1650. The cell culture media were collected, and the IL-2 expression was measured using ELISA-kit and flow cytometry.Lung cancer patient and mouse lung cancer samples were processed for the staining of MUC1-C, PD-L1, CD4, CD8. Immunohistochemical images were captured and the numbers of MUC1-C, CD4, PD-L1, CD8, IFN-γ and Granzyme B positive cells in sections were counted.xenografts on female C57BL/C mice were treatment with 10, 20, 30 mg/kg of evodiamine and xenografts were allowed to grow for over 1 week when tumors were detectable with calipers before treatment by gavages.Animal experiments were performed in accordance with the guidelines by the care and use of laboratory Animals. H1975 5 cells) were intravenously injected into female C57BL/C mice at age 8–10 weeks. Single-cell suspensions of tumors and blood and spleen cells were stained, and cells were acquired using a flow cytometer and analyzed with flowJo software to detect tumor multiplicity in the lung.For the Lewis lung carcinoma model, the Lewis lung carcinoma cells . This study about patients with NSCLC was approved by the ethics committee of Hubei Taihe Hospital.P < 0.05, the results were considered to be statistically significant in this study. Survival rates of mice in our experiment were generated based on the Kaplan–Meier method, statistical significance was determined by the log-rank test, P value < 0.05 is considered statistically significant.All data in this article were expressed as the mean ± SD of three individual experiments. Differences between groups were determined by one-way analysis of variance (ANOVA) by Graph Pad Prism 8 followed by the Bonferroni test to compare all pairs of columns. When 50 value of 5.59 ± 0.88 and 4.53 ± 1.46 μM, respectively, whereas H2228 and HCC827 were less sensitive. H1975 and H1650 are EGFR tyrosine kinase inhibitor resistant lung cancer cells. Evodiamine had much less cytotoxicity in the normal cell line CD19 and BEAS-2B  is an important cytokine with different pleiotropic in the tumor microenvironment, has long been praised as an effector cytokine of anti-cancer immunity and can inhibit cancer development and growth through various mechanisms. However, IFN-γ has also been involved in tumor cell development and transformation. IFN-γ also helps cancer cells evade immune destruction, suggesting that IFN-γ has an existing side effect of promoting tumor. Reports showed that IFN-γcan stimulate the expression of PD-L1 in tumor cells, which bind with PD-1, inhibiting activation, expansion, and effector functions of CD8 evasion . To effe975 Fig. c-f. More975 Fig. g-h.Fig.+ T cell function by increasing IFN-γ and GzmB expression. MUC1-C expression was silenced with shRNA in H1975 and H1650 cells to understand whether MUC1-C could affect T cells proliferation and apoptosis when they were co-cultured with H1975 and H1650 cells. Extended Data Fig. PBMC cells were co-cultured with H1650 or H1975 cells, both expressing high PD-L1. The results show that the apoptosis rate of T cells increased  production  in the spleen, blood and tumor is decreased  than in the patients with early-stage (I) tumors Table. D-E.Tablxenograft and Lewis lung carcinoma model,. We demonstrated that knock out the MUC1-C in H1650 and H1975, both expressing high MUC1-C, can increase IC50 of evodiamine effected on these two cell lines. In our experiment, we observed the inhibitory effect of PD-L1 mRNA and membrane PD-L1 following treatment with evodiamine in IFN-γ stimulation H1650 and H1975. Activation PD-L1 could, through the PD-L1/PD-1 axis could induce the T cells apoptosis in the co-culture system. Evodiamine could downregulate the PD-L1 expression to diminish the apoptosis of T cells. It inhibited MUC1-C expression and potentiated CD8+ T-cell effector function. Evodiamine exhibited anti-tumor activity by elevating of CD8+ T cells in vivo in the Lewis lung carcinoma model. We observed that combination of evodiamine and anti-PD-1 mAb treatment enhanced tumor growth control and survival.In the present study paper, we demonstrated that evodiamine not only significantly inhibits proliferation and increases apoptotic but also shows good anti-tumor activity, which reduces tumor size in H1975 tumor Euodia rutaecarpa, inhibit the proliferation of various tumors and induce tumor cell apoptosis [+ T-cell effector function.The potential impact of Chinese herbal medicines and their ingredients in numerous types of tumor treatment has attracted widespread attention . Extensipoptosis . Evodiampoptosis –45. Evodpoptosis , 44, 46.poptosis . More anpoptosis , 48. A Bpoptosis , 39. Our+ tumor-infiltrating lymphocytes, resulting in reduced in tumor growth [Tumor vaccine fused with soluble PD-1 with the MUC1 gene showed good immunogenicity and anti-tumor effect by enhancing the activation of lymphocytes, and accumulates CD8r growth , 50. MUCr growth . Chimerir growth –54. Consr growth . IFN-γ, r growth . Evodiamr growth .MUC1-C is a transmembrane glycoprotein, which is aberrantly expressed in > 80% Stage IB NSCLC , 55, 56.+ T cells and downregulation of the MUC1-C/PD-L1 axis. This paper corroborates a potential novel mechanism of evodiamine that controls Lung adenocarcinoma cell growth. Our data implies that the rational targeting MUC1-C combined with immunotherapeutic approaches is a potential therapeutic strategy against lung cancer. In our clinical pathological characteristic correlation study, the result indicated that MUC1-C is highly expressed in NSCLC, has high positive rates in female, non-smoker patients with advanced-stage adenocarcinoma. Blocking the MUC1-C/PDL-1 axis is a potential new therapeutic strategy for these subgroup of advanced stage patients.Overall, our results show that evodiamine suppresses NSCLC by elevation of CD8Additional file 1: Extended Data Figure S1. Evodiamine can inhibit growth and induce apoptosis of NSCLC cells. Extended Data Figure S2. Evodiamine inactivates the PD-L1 promoter. Extended Data Figure S3. MUC1-C inhibition can diminish the PD-L1 expression and decrease the apoptosis levels of CD8+T cells. Extended Data Figure S4. Evodiamine potentiates the anti-tumor activity of CD8+ T cells in vivo.Extended Data Figure S5. Combination Evodiamine and PD-1 mAb treatment can enhance tumor growth control and survival of Lewis lung carcinoma model. Extended Data Figure S6. MUC1-C is a potential novel mechanism of evodiamine of tumor inhibition.


Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4 Studies have shown that while high densities of TIL CD8outcomes –3. Carefoutcomes , ectopicoutcomes , 5, 6. Aoutcomes  but haveoutcomes , 9–11. Woutcomes , 5, 12, lonality .+ T cells in regards of TLS-B density. We show that TIL B cells and TIL CD4+ T cells are more highly activated in tumors than in the periphery and that they express key ligand/receptor pairs necessary for B/T interactions and two-way co-stimulation. Moreover, a high density of TLS-B cells is associated with higher frequencies of activated CD4+ T cells and lower frequencies of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the intratumor CD4+ T cell compartment. High densities of TLS-B cells together with low densities of FoxP3+ Tregs in NSCLC tumors consistently identified the group of patients with the best clinical outcome.In light of these findings, we used extensive gene expression profiling and flow cytometry for an integrative analysis of the phenotypes of TIL B cells and TIL CD4+ T cell-mediated immune responses.Overall, these results suggest that TLS-B cells promote the development of protective CD4n = 26) or perform flow cytometry analyses  .This retrospective study examined formalin-fixed, paraffin-embedded (FFPE) primary NSCLC samples from 538 patients who underwent surgery without any neoadjuvant chemoradiotherapy between 2001 and 2005 at Hôtel Dieu Hospital in Paris, France . We alsonalyses) . PatientFFPE NSCLC blocks were selected, and sections were stained as previously described , 12, 14,Cells from the entire tumor section were quantified with Calopix® software (Tribvn). Because TLS-B cells were highly aggregated , their d+ TLS-B cells (mm2)/Total tumor area (mm2)]*100. CD4+ T cells and [PD-1+ TIGIT+] CD4+ T cells  and the phenotype of fresh intratumor CD4 T cells , and cor T cells .+ T cells   , we furtng FoxP3  that wash tumors . A signir sample , and to  density . The perh tumors . This obh tumors .+ T cell compartment of tumors with a low density of TLS-B cells.These results demonstrate that Tregs are most frequent in the CD4+ TLS-B cells was next investigated in a retrospective cohort , peaked at stage II  but no longer existed at stages III-IV   . The pro 0.1649) .+ FoxP3+ Tregs .Next, we evaluated the prognostic value of CD3In univariate Cox regression analysis, patients' age (HR = 2.239), pTNM stage (HR = 2.841), and high Treg density (HR = 1.617) were each significantly associated with poor OS, and high TLS-B cell density (HR = 0.606) with long-term OS . MultivaTaken together, these data show that high TLS-B cell density significantly and positively affects survival of NSCLC patients, and its evaluation makes it possible to better stratify patient prognosis, especially when combined with low Treg cell density.+ T cells  that directly contributes to IFN-γ and IL-2 promoter activities  co-signaling receptor family. After activation through a productive TCR-MHC-peptide interaction, 4-1BB, OX40, and GITR are induced and synergize with the TCR and CD3 molecules to promote cell cycle progression, survival, and cytokine production by T cells , which inhibits the proliferative capacity of tumor-infiltrating effector T cells when expressed by cancer-associated fibroblast-subset 1 (CAF-S1) conditioned-Treg in triple-negative breast cancer . Samples from healthy volunteers were obtained at the Etablissement Français du Sang . The patients/participants provided their written informed consent to participate in this study.M-CD-N and SH conceptualized the research. CG, PD-M, SK, JB, HK, LL, JG, and FL performed research investigation. AS-G, DG, NG, PV, ML, DD, and MA provided resources (fresh human specimens and patient enrollment). CG and M-CD-N performed formal analysis of the data. CG wrote the initial draft. M-CD-N, KS, SH, and J-LT revised and edited the manuscript. M-CD-N performed funding acquisition. All authors read and approved the final version of the manuscript.DG and AS-G received fees from Medtronic company for presentations not in relation with this topic. DG is consultant for an instrument manufacturer . KS and SH are full-time employees of AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The microRNA, miR-139-5p, has been proved to play important roles in regulating tumor progression, including prostate cancer, osteosarcoma, esophageal cancer, and so on, but its correlation of hepatocellular carcinoma (HCC) still remains unclear. Here we found that hsa-miR-139-5p (miR-139-5p) was decreased in HCC samples compared with normal liver tissues, and a lower expression of miR-139-5p was connected to a poorer prognosis. Mechanism study indicated that a decreased/increased miR-139-5p could increase/decrease HCC cells invasion and proliferation capacities via increasing SLITRK4 expression, what’s more, the reverse assays also confirmed the conclusion when we knocked down SLITRK4 in the miR-139-5p low-expression cells. Luciferase assay confirmed that miR-139-5p could directly bind to the 3′UTR of SLITRK4 mRNA to regulate its expression. Together, these findings show the importance of miR-139-5p/SLITRK4 pathway in HCC growth and progression and may provide new targets for us to better arrange the progression of HCC. Hepatocelluar carcinoma (HCC) is one of the main health problems faced by the public across the word. According to Cancer Statistics, HCC has become the second leading cause of cancer in the world, especially in Africa and Asia . What’s MicroRNAs (miRNAs) belong to the family of non-coding RNAs with 20–24 nucleotides in length. Usually, miRNAs can silence the target genes by post-transcriptional regulation by binding to the 3′ untranslated region (UTR) of target genes , and dysSLIT and NTRK like family member 4 (SLITRK4), located at Xq27.3, belongs to a six-member family of synapse organizers that control excitatory and inhibitory synapse formation by forming trans-synaptic adhesions with LAR receptor protein tyrosine phosphatases (PTPs) ,16. SLITHere, our study showed that the miR-139-5p expression was decreased in HCC tumor samples, which could increase HCC invasion and proliferation capacity by increasing SLITRK4 expression. Thus, we find a new pathway between miR-139-5p and HCC progression, and may provide novel targets for better therapies for HCC.http://ngs.ym.edu.tw/ym500v2/index.php), OncomiR database (http://www.oncomir.org/oncomir/search_target_miR.html), Kaplan Meier plotter database  and GEPIA database (http://gepia.cancer-pku.cn/index.html) were used to analyze the clinical correlations and significance of miR-139-5p and SLITRK4 in normal and HCC samples. All the clinical data were gotten from these databases and used according to the instructions and rules of the databases.The YM500v2 database  with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% glutamine. All cells were cultured in a 37°C temperate box with 5% (v/v) CO5 HCC cells were seeded into 8-μm transwell chamber (Corning Life Science) with 100 μl diluted Matrigel (BD Corning) coated first in a serum-free medium. The bottom chamber was filled with 700 μl medium (10% FBS). After cultured for 24 h, we collected the transwell chamber for fixation and dyeing of the invaded cells, then observed and counted the cells under the microscope. Each sample was designed in three duplicate chambers and several pictures under different horizons were taken for analysis.A total of 1 × 103 HCC cells were seeded into 24-well plate, and when all the cells adhered to the plate, we marked it as ‘day 0’. We collected the cells of ‘day 0’, ‘day 1’, ‘day 3’, ‘day 5’ for MTT assay. First, we added 500 μl 0.5 mg/ml MTT solution into each hole of the plate and cultured in a 37°C temperate box with 5% (v/v) CO2 condition for 2–4 h, then we removed the solution and added 200–500 μl DMSO into the plate. After shaking for 20 min on a shaker, the absorbance of the cells at 570 nm was measured. Each sample was designed in three duplicate chambers and a curve connecting the value of each day was drawn to analyze the proliferation capacity.A total of 5 × 10About 30 μg proteins were loaded into 10% SDS/PAGE gel and then transferred onto a PVDF membrane. After incubated with the primary antibody in a 4°C condition for overnight and secondary antibody at room temperature for 1–2 h, the membrane was visualized with ECL system. The SLTRK4 (ab67315), PCDH7 (ab139274) and GAPDH (6C5) antibodies were purchased from Abcam company. UGT2A3 (PA5-48900) antibody was purchased from Thermo Fisher Scientific company.About 2 μg of RNA was reverse transcribed into cDNA, and the relative expression was calculated by a Bio-Rad CFX96 system. U6 primer was used as internal control to normalize the relative expression. The primers sequences used in the study were as follows: has-miR-139-5p: TCTACAGTGCACGTGTCTCCAGT; U6: Forward: CTCGCTTCGGCAGCACA, Reverse: AACGCTTCACGAATTTGCGT. All the primers were ordered from IDT Company.The pLKO.1-miR-139-5p (oemiR-139-5p), pLKO.1-shSLITRK4 (shSLITRK4), pWPI-SLITRK4 (oeSLITRK4) plasmids, the psPAX2 packaging plasmid and pMD2.G envelope plasmid were transfected into HEK-293T cells using the standard calcium chloride transfection method for 48 h to get the lentivirus soup. The lentivirus soups were collected and concentrated by density gradient centrifugation and used immediately or frozen at −80°C for later use. Lentivirus soups were added into the medium until the cells were 60% confluent. Polybrene  was added to improve tansfection efficiency. Twenty-four hours later, the medium was refreshed and transfected with lentivirus soups again. The transfected cells were used for further experiments after checked the tansfection efficiency.About 1000 bp of SLITRK4 3′UTR was chosen and the potential binding site of miR-139-5p was obtained by using the Targetscan and Genome databases. Based on which, we constructed the wild-type and mutant (the binding site was replaced by GGATCCA) SLITRK4 3′UTR psiCHECK-2 plasmids. Then, the HCC cells were transduced with the wild-type/mutant plasmids and pLKO/oemiR-139-5p or control/miR-139-5p inhibitor . After cultured for 48 h, the cells were collected and the luciferase activity was measured by a dual luciferase reporter assay according to the protocol.The miR-139-5p inhibitor (MH11749) was purchased from Thermo Fisher Scientific Company. The oemiR-139-5p (pLKO-miR-139-5p) sequence was as follows: Forward: CCGGTCTACAGTGCACGTGTCTCCAGTTTGGATCCGACTGGAGACACGTGCACTGTAGATTTTTG; Reverse: AATTCAAAAATCTACAGTGCACGTGTCTCCAGT CGGATCCAAACTGGAGACACGTGCACTGTAGA.F1: CAGTAATTCTAGGCGATCGC GTCATGTAATCTTACTTCTTR1: AGATATTTTATTGCGGCCAGCTACTTACTGATAAATTCACTF2:CTGCAAAGATGTGTCAAGTAR2: TACTTACTGATAAATTCACTGGATCCATAATGAGACACCCTTGGAAAThe wild type/mutant plasmid sequences were:t test and the mean values of more than two groups were compared with one-way ANOVA. We thought it as significant difference when P≤0.05.All statistical analysis were conducted by SPSS 22.0 software, and the data were shown as mean ± SD. Differences in mean values between two groups were analyzed by two-tailed Student’s http://ngs.ym.edu.tw/ym500v2/index.php). The result showed that the expression of miR-139-5p in liver ranked the third among the overall 21 organs (http://www.oncomir.org/oncomir/search_target_miR.html). The result showed that the expression of miR-139-5p was lower in HCC samples than that in normal tissues , and the result proved that a lower expression of miR-139-5p meant a lower survival rate in HCC patients to check SLITRK4 clinical significance in HCC samples, and the data showed that SLITRK4 expression was increased in HCC samples and a higher SLITRK4 meant a lower survival rate in HCC patients  A. We thepatients D,E.To assess the function of SLITRK4 under the regulation of miR-139-5p in HCC, we added miR-139-5p inhibitor and knocked down SLITRK4 in HCC cells, and the reverse assays showed that knocking down SLITRK4 could partly reverse miR-139-5p inhibitor’s function to HCC invasion and proliferation, both in HCC SK-HEP-1 and HA22T cells A–D. WhatTogether, the results from http://www.targetscan.org/vert_72/) and Genome (http://genome.ucsc.edu/cgi-bin/hgGateway) databases to find out the binding sites of miR-139-5p on the 3′UTR of SLITRK4 mRNA and constructed a 1000 bp wild-type and mutant sequences  is the primary and main type of liver cancer, in 2012, there were 78,250,000 people diagnosed with HCC and 74,550,000 people died of this cancer across the word, and among which, more 50% of the newly diagnose patients and deaths were Chinese . ChronicThe α-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-2 (PIVKA-II) are usually used as diagnostic markers for HCC at early stage, but in clinical practice, many HCC patients went late stage when first diagnosed, and HCC patients usually have high post-operative recurrence, many studies have been conducted to find new or more related biomarkers to better predict HCC. He et al.  concludeEvery year, the government has provided millions of money to the studies of HCC, and sorafenib and regorafenib have made great contributions for the therapy of HCC. What’s more, many studies have focused the mechanisms of HCC progression and metastasis . Our stumicroRNAs (miRNAs) are short (20–24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. MiR-139-5p, located on chromosome 11q13.4, has been reported to play important roles in many cancers . In our in vitro study also proved the importance of SLITRK4 in HCC progression. All of this highlight the importance of SLITRK4 in HCC biological process, and target this SLITRK4 related signaling may provide a new choice for the treatment of HCC.SLITRK4 gene can encode a transmembrane protein, which belongs to the family of SLITRK. The SLITRK4 family members have been reported to include two N-terminal leucine-rich repeat domains that are similar to those in SLIT, the axonal growth-controlling protein, as well as C-terminal regions similar to neurotrophin receptors. SLITRK4 is expressed in many tissues, among which adrenal and brain tissues rank the first two. Until now, only a few studies have reported the function of SLITRK4, many of them are related to nervous system disease ,19,34,35In conclusion, our study showed that the miR-139-5p expression was decreased in HCC tumor samples, which could increase HCC invasion and proliferation capacity by increasing SLITRK4 expression. Thus, we find a new pathway between miR-139-5p/SLITRK4 and HCC progression, and target this newly pathway may provide us better therapies for HCC.


Despite the activation of autophagy may enable residual cancer cells to survive and allow tumor relapse, excessive activation of autophagy may eventually lead to cell death. However, the details of the association of autophagy with primary resistance in hepatocellular carcinoma (HCC) remain less clear. In this study, cohort analysis revealed that HCC patients receiving sorafenib with HBV had higher mortality risk. We found that high epidermal growth factor receptor (EGFR) expression and activity may be linked to HBV-induced sorafenib resistance. We further found that the resistance of EGFR-overexpressed liver cancer cells to sorafenib is associated with low activity of AMP-activated protein kinase (AMPK) and CCAAT/enhancer binding protein delta (CEBPD) as well as insufficient autophagic activation. In response to metformin, the AMPK/cAMP-response element binding protein (CREB) pathway contributes to CEBPD activation, which promotes autophagic cell death. Moreover, treatment with metformin can increase sorafenib sensitivity through AMPK activation in EGFR-overexpressed liver cancer cells. This study suggests that AMPK/CEBPD-activated autophagy could be a potent strategy for improving the efficacy of sorafenib in HCC patients. Sorafenib is a multi-kinase inhibitor that mainly targets Raf kinases and receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR)-2/3, platelet-derived growth factor receptor (PDGFR)-β, FMS-like tyrosine kinase 3 (Flt3), and c-Kit (CD117) /hepatitis C virus (HCV) has been suggested to support viral replication and contributes to HCC progression  is a transcription factor that responds to various external stimuli, including the proinflammatory cytokines IL-1β and TNFα  is a current first-line pharmacological treatment for type 2 diabetes. Some studies have further demonstrated that metformin can induce cell arrest and promote cell death  and National Health Insurance Research Database (NHIRD) to identify diagnosis of HCC and sorafenib prescription  code 155.0 were used to identify patients diagnosed with HCC between 2012 and 2015 from the TCR database. Information on HBV or HCV infection were obtained for the period from 12 months before until 12 months after HCC diagnosis based on ICD-9-CM diagnosis codes: HBV  and HCV . Patients with a previous cancer history, a lack of clear demographic and tumor information or aged <18 years were excluded. Finally, a total of 6,628 HCC patients were enrolled in this analysis. For the usage of sorafenib, all patients were reimbursed without co-payment by NHI according to the criteria BCLC advanced stage that were not amenable to either surgical resection or locoregional therapy and Child–Pugh class A liver functional reserve. The prescription of sorafenib is 800 mg (200 mg/tablet) for 2 months. The application needed to be re-evaluated every 2 months for next term of sorafenib usage with imaging evidence showing no disease progression.p < 0.05 was considered statistically significant.The categorical variables were presented as frequency with percentage, and the difference between patients with HBV diagnosed and those without was compared using Pearson's chi-square test. The 1-year mortality risk for HCC patients with different hepatitis B/C virus was estimated using Cox proportional regression analysis adjusted with age, gender, HCC diagnosed to start sorafenib, dosage of sorafenib, comorbidities, and additional therapy after sorafenib such as TACE, RFA, radiation, hepatectomy, and liver transplantations. The stratified analysis was also implemented to investigate the mortality risk among the different duration of sorafenib used. In addition, the estimation of different follow-up period mortality risk was considered. The predicted survival curves were plotted using the results of above Cox regression analysis with adjusted confounding factors. SAS 9.4 for Windows  was used for all statistical analyses. All statistical tests were 2-sided, and 2.The human hepatocellular carcinoma cell lines Huh7 and Hep3B were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 units/ml penicillin at 37°C and 5% COThe virus was produced from Phoenix Ampho cells using Mirus Bio TransIT-2020 and cotransfected with various short hairpin RNA (shRNA) expression vectors in combination with pMD2.G and psPAX2 vectors and the pLKO.1-shRNA expression vectors. The short interfering RNA sequences targeting LacZ, CEBPD, and AMPK were subcloned into the lentiviral expression vector pLKO.1. The short interfering RNA sequences are as follows: shLacZ (shZ): 5′-CCGGTGTTCGCATTATCCGAACCATCTCGAGATGGTTCGGATAATGCGAACATTTTTG-3′; shCEBPD (shD): 5′-CCGGGCCGACCTCTTCAACAGCAATCTCGAGATTGCTGTTGAAGAGGTCGGCTTTTT-3′; shAMPKα (shKα1): 5′-CCGGTGATTGATGATGAAGCCTTAACTCGAGTTAAGGCTTCATCATCAATCATTTTT-3′; shAMPKα (shKα2): 5′-CCGGCAACTTTACCTGGTTGATAACCTCGAGGTTATCAACCAGGTAAAGTTGTTTT-3′. The expression vectors and shRNAs were obtained from the National RNAi Core Facility located at the Genomic Research Center of Institute of Molecular Biology, Academia Sinica, Taiwan.Human CEBPD reporter was constructed in our lab  of sorafenib for 48 h or with the combination of 2.5 μM sorafenib and 5 mM metformin for 48 h. The experimental cells were incubated with diluted MTT reagent  at 37°C for 3.5 h. The samples were then measured spectrophotometrically at 595 nm by an ELISA plate reader.Huh7 and Hep3B cells were seeded 54 cells were analyzed by FACScan cytometry . The data were determined by three independent experiments.Huh7 and Hep3B cells were treated with sorafenib for 48 h. Treated and control cells were harvested, washed twice and re-suspended in 500 μl of PBS plus Annexin V-FITC and PI in dark for 15 min at room temperature. The degree of apoptosis was determined as the percentage of cells positive for Annexin V-FITC/PI. For each sample, at least 1 × 10 The pEGFP-LC3 plasmid was a gift obtained from Dr. Tamotsu Yahsimori and Noboru Mizushima  in 0.2 ml PBS were inoculated subcutaneously into the right flank of the mice. After 14 days, when macroscopic tumors (50–100 mm3) had formed, animals were placed randomly into four groups (n = 5 per group) as follows: (1) the control group, which received identical volumes of vehicle; (2) the sorafenib treatment group, which was treated with sorafenib at doses of 15 mg/kg/day; (3) the metformin treatment group, which was treated with 250 mg/kg/day metformin; and (4) the combined treatment group, which was injected with sorafenib combined with metformin. Treatment was given to all groups intraperitoneally every day for 4 weeks. Animal weight and tumor dimensions were measured every 4 days with calipers, and tumor volumes were estimated using two-dimensional measurements of length and width and were calculated with the formula: [l × (w)2] × 0.52, where l is length and w is width.Male, 6-week-old NOD/SCID mice were obtained from the Laboratory Animal Center of National Cheng Kung University, Tainan, Taiwan. Hep3B cells  or without HBV/HCV   . After a= 2,113) . In addi= 2,113) , and the= 2,113) , suggest= 2,113) . Further= 2,113) , suggestHBV-negative) and Hep3B (derived from HBV-infected liver) cells. The results showed that, in contrast to that in Huh7 cells, the activity of ERK1/2  was sustainedly activated in Hep3B cells, and there was no further effect following sorafenib treatment , a potential biomarker for sorafenib response, was examined in Huh7 -transfected Huh7 cells  and phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways  are potent agonist ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). They are also thought to exert some of their anti-diabetic effects through AMPK activation in a variety of tissues, including skeletal muscle .The studies involving human participants were reviewed and approved by Institutional Review Board of Chi-Mei Medical Center in Taiwan (IRB: 10702-E04). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of College of Medicine, National Cheng Kung University .H-YLa, H-HT, C-FL, and J-MW: conceptualization. H-YLa, H-HT, C-JY, L-YH, C-CY, C-HH, H-YLi, and F-WC: methodology and formal analysis. H-YLa, H-HT, C-JY, L-YH, C-CY, and C-HH: investigation. H-YLa, H-HT, C-JY, L-YH, C-CY, C-HH, C-FL, and J-MW: resources. H-YLa and H-HT: validation, visualization, and writing–original draft. H-YLa and J-MW: writing–review & editing. J-MW: funding acquisition and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species:  The attached oligosaccharide is subsequently processed in the ER and Golgi, resulting in a wide variety of structures which can differ greatly between genera  NaOCl solution prior to being sown to half-strength Murashige-Skoog (MS) plates containing 1% (v/w) sucrose and 0.8% (w/v) agar. Plants were grown under long-day conditions (16 h light/ 8 h dark) at 22°C and transferred to soil 7 days after sowing. The galt1 fut13 double knockout mutant was obtained by crossing previously described lines galt1 (N871760) and fut13 (N567444)  buffer . To isolate Lewis A bearing glycoproteins, we used the Lewis A-specific JIM84 antibody  to a final concentration of 3.5 ng/μL, incubated overnight at 37°C and peptide fragments were isolated on the next day via centrifugation. The supernatant was subjected to a final clean-up step using a C18 Hypersep cartridge (Thermo Fisher Scientific). The cartridge was washed three times with 65% (v/v) acetonitrile in 80 mM ammonium formate buffer pH 3, equilibrated in the same buffer and loaded with the eluted peptides in 100 mM NH4HCO3 buffer pH 7.8. After washing three times, peptides were eluted with 65% acetonitrile in pH 3 buffer and vacuum dried.For isolation of Lewis A bearing glycoproteins, leaves and stems of 5-week-old N. benthamiana, a recently published database was used , the following settings were used: reversed sequences no; check parent ions for charges 1, 2, and 3 yes; models found with peptide log e lower −1 and proteins log e lower −1; residue modifications: oxidation M, W, and deamidation N, Q; isotope error was considered; fragment type was set to monoisotopic; refinement was used with standard parameters; fragment mass error of 0.1 Da and ±7 ppm parent mass error; fragment types b and y ions; maximum parent ion charge of three; missed cleavage sites allowed was set to two; semi-cleavage yes.Analysis of isolated Lewis A bearing glycoproteins was conducted using nano-LC-ESI-MS as previously described  and At5g15630_2R (5′-TATAAGATCTTCACCATATTGAGATGAATAGGAGA-3′), digested with XbaI/BglII and ligated into XbaI/BamHI-digested p117  and g6145_R2(5′-TATAGGATCCCTAATAGAACACAGAAAAGATTGCA-3′), digested with XbaI/BamHI and ligated into XbaI/BamHI-digested p117. For expression of CEBIP, a codon-optimized version for N. benthamiana lacking the endogenous signal peptide coding sequence (bases 1–84) was ordered from GeneArt (Thermo Fisher Scientific). The DNA sequence was amplified using primers STRINGS_9F (5′-CTTCCGGCTCGTTTGTCTAGA-3′) and STRINGS_2R (5′-AAAAACCCTGGCGGGATCC-3′), digested with XbaI/BamHI and ligated into XbaI/BamHI-digested p117.For expression of identified proteins in N. benthamiana, syringe-mediated leaf infiltration of 5-week-old plants was used. Agrobacterium tumefaciens strain UIA143 -ethanesulphonic acid (MES), 2 mM Na3PO4.12H2O, 0.1 mM acetosyringone). p29-Fc-KOR1 was additionally co-infiltrated with silencing suppressor p19  and snap-frozen. For stable expression in A. thaliana, plants were transformed using the floral dip method  according to the manufacturer's instructions. For deglycosylation experiments with Peptide-N-glycosidase F , proteins purified from ΔXT/FT-N. benthamiana , ethanol , chloroform , and acetone . Remaining cell wall residues were weighed, and percentage of full stem weight calculated. For quantification of cellulose, half of the cell wall residue fraction was incubated in trifluoroacetic acid (TFA) for 3 h with shaking in-between to remove hemicellulose. After washing with acetone, the remaining pellet was dried and weighed. Updegraff-reagent  sulphuric acid, incubated, H2O added and centrifuged. The supernatant was diluted 1:10, mixed with anthrone reagent in sulphuric acid in a 96 well plate, heated to 80°C for 30 min and subsequently absorption was measured at 595 nm using a plate reader . Cellulose content in the samples was calculated based on a glucose–standard curve and percentage of cellulose per cell wall residues calculated. For determination of lignin, the second half of the cell wall residue pellet was mixed with 25% (v/v) acetyl-bromide solution in acetic acid, heated for 3 h at 50°C and centrifuged. The supernatant was diluted 1:20 in a solution containing 0.4 M NaOH and 18.75 mM hydroxylamine hydrochloride in acetic acid and absorption measured at 280 nm . Lignin content was then calculated as percentage of cell wall residues.Cell wall constituents were quantified as previously described , plates containing 150 mM NaCl  or plates containing 12% (w/v) PEG6000 corresponding to a water potential of −0.5 MPa for simulation of drought stress via the PEG infusion method , Endoglucanase 25  and Protein trichome-birefringence like 3. All these proteins have numerous potential N-glycosylation sites and a predicted localization in the secretory pathway. Interestingly, many of the identified proteins have a proposed function in secondary cell wall biosynthesis. In addition, we detected Laccase 17, Probable inactive purple-acid-phosphatase and Protein trichome-birefringence-like 33 in siliques as well as O-fucosyltransferase family protein SUB1 in stems of A. thaliana. The list obtained from O. sativa comprises proteins of various functions. We detected two defense related proteins, Chitin elicitor-binding protein (CEBIP) and Germin-like protein 2–4. Furthermore, Dirigent protein, which is involved in biosynthesis of secondary metabolites, Os02g0615800, a receptor kinase, Beta-Ig-H3 domain-containing protein, a cell surface adhesion protein, and Os08g0503200, a glycerophosphodiester phosphodiesterase. In N. benthamiana . We found only one SNP with a predicted high impact, an alteration in the nucleotide sequence leading to the formation of a premature stop codon in the FUT13 gene. This SNP is present in 55 accessions and results in the expression of a truncated FUT13 protein where the two C-terminal amino acids (“GV”) are missing. Since there is some amino acid sequence variation at the C-terminus of FUT13 from different species we consider it unlikely that the truncation completely abolishes FUT13 function. In addition, polymorphisms were detected in the FUT13 gene which might have an impact on FUT13 activity. As GALT1 catalyzes the rate limiting reaction of Lewis A biosynthesis, we decided to investigate polymorphisms in the GALT1 gene in more detail. We found only one accession, IP-Orb-10, containing a premature stop codon instead of the codon encoding the last amino acid W643. galt1-fut13 and galt1  either only a low number of specific glycoproteins are modified with Lewis A structures or (2) Lewis A structures are present on various glycoproteins in the trans-Golgi, the plasma membrane and the apoplast, but occur in so small quantities that we could identify only the most abundant glycoproteins or the ones carrying complex N-glycans with high amounts of Lewis A, respectively. Based on these possibilities we cannot rule out that we only enriched a certain population of Lewis A bearing glycoproteins using the described purification approach. Overall, the information about Lewis A bearing glycoproteins published in literature is rather scarce. In a recent study investigating N-glycan microheterogeneity in, among others, inflorescence stems of A. thaliana  and may also affect the FUT13 activity. While the truncated FUT13 sequence is found in 55 different accessions, the T294K polymorphism is only found in one other line IP-Vis-0. In the GALT1 gene from IP-Vim-0 four polymorphisms are present that lead to changes of the amino acid sequence. Two of the changes (Q243L and K253N) are also found in Lewis A-positive lines such as MNF-Pin-39 and therefore do not inactivate GALT1. The other two polymorphisms are present in a conserved sequence motif that is present in the catalytic domain of CAZy GT31 family members (Qu et al., GPGYIVSRDIA, it is GPGNIVSRDIE in IP-Vim-0. Both mutations very likely abolish the enzymatic activity of GALT1 and lead to the absence of Lewis A bearing N-glycans in IP-Vim-0. This line has been defined as a relict, meaning high genomic divergence from geographically proximal accessions (Alonso-Blanco et al., The Lewis A trisaccharide on complex plant N-glycans is an evolutionarily conserved modification throughout the plant kingdom (Fitchette et al., The original contributions presented in the study are included in the article/GB, EV, and RS designed the experiments. GB and DM conducted the experiments. GB, DM, EV, FA, and RS analyzed the results. GB and RS wrote the paper. All authors have made a substantial and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-glycosylation in eukaryotic cells. A K42E point mutation in the DHDDS gene causes an autosomal recessive form of retinitis pigmentosa (RP59), which has been classified as a congenital disease of glycosylation (CDG). We generated K42E Dhdds knock-in mice as a potential model for RP59. Mice heterozygous for the Dhdds K42E mutation were generated using CRISPR/Cas9 technology and crossed to generate DhddsK42E/K42E homozygous mice. Spectral domain-optical coherence tomography (SD-OCT) was performed to assess retinal structure, relative to age-matched wild type (WT) controls. Immunohistochemistry against glial fibrillary acidic protein (GFAP) and opsin (1D4 epitope) was performed on retinal frozen sections to monitor gliosis and opsin localization, respectively, while lectin cytochemistry, plus and minus PNGase-F treatment, was performed to assess protein glycosylation status. Retinas of DhddsK42E/K42E mice exhibited grossly normal histological organization from 1 to 12 months of age. Anti-GFAP immunoreactivity was markedly increased in DhddsK42E/K42E mice, relative to controls. However, opsin immunolocalization, ConA labeling and PNGase-F sensitivity were comparable in mutant and control retinas. Hence, retinas of DhddsK42E/K42E mice exhibited no overt signs of degeneration, yet were markedly gliotic, but without evidence of compromised protein N-glycosylation. These results challenge the notion of RP59 as a DHDDS loss-of-function CDG and highlight the need to investigate unexplored RP59 disease mechanisms.Dehydrodolichyl diphosphate synthase (DHDDS) is required for protein  Relatsylation ,7. DHDDSsylation ,9. This proteins . The monproteins ,12. In ain vitro , as wellin vitro .DhddsK42E/K42E) of RP59—since K42E is the most prevalent point mutation in the RP59 patient population [N-glycosylation underlies the retinal dysfunction and degeneration observed in human RP59. Herein, we present a description of the generation and initial characterization of the phenotypic features of the DhddsK42E/K42E mouse model. Surprisingly, although we expected to observe an early onset, progressive, and potentially severe retinal degeneration, this was not the case. The retina appeared histologically intact and normal according to spectral domain optical coherence tomography (SD-OCT) analysis for up to at least one year of age. However, there was evidence of gliotic reactivity  immunostaining), despite the lack of obvious neuronal degeneration or cell death/loss. Also, despite the homozygous mutation in Dhdds, we found no evidence of compromised protein N-glycosylation in mutant mouse retinas.In the present study, we created a DHDDS K42E homozygous knock-in mouse model  mice were generated on a C57Bl/6J background by Applied StemCell . Briefly, CRISPR guide RNA (5′-TCGCTATGCCAAGAAGTGTC-3′ with PAM site AGG) was generated using in vitro transcription and was used to create a double strand break in the murine Dhdds locus to promote introduction of a single-stranded oligodeoxynucleotide (SSO) carrying the K42E mutation and a second silent DNA polymorphism to eliminate the PAM recognition site required for cleavage by CAS9 (5′-ATTATCTGTTCTCTTCTACAGGCTGGCCCAGTACCCAAACATATCGCGTTCATAATGGACGGCAACCGTCGCTATGCCAAGGAGTGTCAAGTGGAGCGCCAGGAGGGCCACACACAGGGCTTCAATAAGCTTGCTGAGGTGGGTGCGGGTGACAGAGCCTAGA-3′). Mouse zygotes were injected with 100, 100, and 250 ng/μL of Cas9 enzyme, guide RNA, and SSO, respectively, which were then transferred into pseudo pregnant CD-1 females. Three potential founder (F0) pups were identified out of 13 mice tested, and an F0 founder was verified by DNA sequence analysis. Sequence-validated heterozygous (DhddsK42E/+) mice were crossed to generate homozygous (DhddsK42E/K42E) mice, as confirmed by PCR and DNA sequencing (see below). C57Bl/6J wild type (WT) mice, age- and sex-matched, were used as controls. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham. All animals were maintained on a standard 12/12 h light/dark cycle (20–40 lux ambient room illumination), fed standard rodent chow, provided water ad libitum, and housed in plastic cages with standard rodent bedding.Heterozygous (K42E/+) DhddsK42E/K42E mice. For initial verification of the knock-in, PCR products were sequenced in the UAB Heflin Center for Genomic Sciences. The presence of the knock-in sequence was confirmed in subsequent generations by restriction enzyme digestion with StyI, which cleaves the knock-in allele only (data not shown). Knock-in alleles were independently verified by Transnetyx, Inc.  using proprietary technology. While the analysis was set up to recognize and differentiate the knock-in mutation and the PAM site polymorphism, only the knock-in mutation was maintained in all subsequent breeding.PCR primers were designed that spanned the targeted region  amplifying a 292 bp segment of DNA in both WT and In vivo retinal imaging was performed as previously described in detail by DeRamus et al. [DhddsK42E/K42E and WT mice at postnatal day (PN) 1 , 2 , 3 , 8 , and 12 months (mos)  to assess retinal structure. Layer thicknesses were determined manually using Bioptigen InVivoVue® and Bioptigen Diver® V. 3.4.4 software and the data were analyzed and graphed using Microsoft Excel software. s et al. , using a®-488 conjugated anti-mouse IgG, AlexaFluor®-568 conjugated anti-rabbit IgG; Thermo Fisher Scientific, Waltham, MA, USA; 1:500 dilution in TBST). Sections were then counterstained with DAPI and cover slipped with anti-fade mounting medium  and viewed with a Leica TCS SPEII DMI4000 scanning laser confocal microscope (Leica Biosystems). Images were captured using a 40X oil immersion (RI 1.518) objective under normal laser intensity (10% of laser power source), arbitrary gain (850 V) and offset (–0.5) values, to optimize signal-to-noise ratio. Digital images were captured and stored as TIFF files on a PC computer.Procedures utilized for fixation, O.C.T. embedment, and sectioning of mouse eyes were as described in detail previously by Ramachandra Rao et al. . In brie®-488 conjugated streptavidin  and AlexaFluor®-647-conjugated peanut agglutinin , with or without pre-treatment  with peptide:N-glycosidase F . Sections were DAPI-stained and mounted using Vectashield mounting media, and digital images obtained using scanning laser confocal microscopy as described above [Paraformaldehyde-fixed eyes (as described above) were processed for paraffin embedment. Paraffin sections of mouse eyes were then incubated (45 min at room temperature) with biotinylated Concanavalin-A , followed by incubation with AlexaFluored above .K42E knock-in mice were generated commercially using CRISPR-Cas9 technology. The K42E knock-in mutations in both heterozygous and homozygous mice were confirmed by DNA sequence for one of the heterozygous F0 founder mice, which is shown in in vivo. Qualitative SD-OCT images obtained from wild type (WT) and DhddsK42E/K42E mice are presented in SD-OCT provides a non-invasive means of assessing retinal morphology DhddsK42E/K42E mice, compared to age-matched WT control littermates. The data are shown both with respect to outer nuclear layer (ONL) thickness (yellow and gray lines) as well as total neural retina thickness (blue and orange lines) as a function of distance from the optic nerve head  along the vertical meridian, for both the inferior and superior hemispheres. No differences in these quantitative metrics of retinal morphology were observed with respect to genotype, consistent with the representative OCT images shown in We used SD-OCT to perform quantitative analysis of retinal morphology to compare ocular tissue layer thicknesses in WT and knock-in mice. DhddsK42E/K42E mouse eyes at PN 2 months of age, probing with antibodies against GFAP  and a C-terminal epitope of rod opsin . As shown in DhddsK42E/K42E mice exhibited extensive, robust anti-GFAP labeling in a radial pattern. This extended throughout the inner retinal layers to the outer plexiform layer (OPL), in addition to intense labeling along the vitreoretinal interface. The latter results are indicative of massive gliotic activation, which is remarkable considering the lack of overt retinal degeneration or loss of retinal neurons . Anti-opsin immunolabeling (pseudocolored green) was comparable in both WT and DhddsK42E/K42E retinas. Notably, the label was confined to the OS layer; there was no mislocalization of opsin to the plasma membrane of the cell in the IS or ONL layer—unlike what is often observed in degenerating photoreceptor cells in various animal models—suggesting normal trafficking of opsin to the outer segment, and consistent with a lack of overt photoreceptor degeneration. It is worth noting that the green labeling in a few cells in the inner retina in We performed immunohistochemical analysis on frozen sections of fixed, O.C.T.-embedded WT and N-linked oligo-saccharides of glycoproteins contain alpha-linked mannose residues as constituents, which are cognate ligands for the lectin concanavalin A (Con A) [DhddsK42E/K42E mice, and a way to directly test the current hypothesis that RP59 is driven by lack of glycosylation. This is because the synthesis of oligosaccharide chains in cells and tissues obligatorily depends upon the presence of Dol-PP and Dol-P (which requires upstream DHDDS activity). Furthermore, N-linked oligosaccharide chains are selectively susceptible to hydrolysis by peptide:N-glycosidase F (PNGase-F) [DhddsK42E/K42E mice at PN six months of age, with and without pre-treatment with PNGase-F. The results are shown in The  (Con A) . Hence, NGase-F) ; hence, N-linked glycoproteins are present throughout the retina, being notably enriched in photoreceptor cells and the synaptic endings of neurons . Hence, the inner and outer segment layers , including the glycoconjugate-rich interphotoreceptor matrix (IPM), as well as the inner and outer plexiform layers  were robustly labeled with fluor-tagged ConA in untreated WT retinal sections , rather than N-linked, and their synthesis is not dolichol-dependent in mammalian cells [DhddsK42E/K42E retinas vs. WT controls, nor effects of PNGase-F treatment on PNA binding, either with regard to labeling intensity or distribution. These expectations were realized, as illustrated in DhddsK42E/K42E and control retinas exhibited comparable distribution of PNA-positive cone matrix sheaths, suggesting persistence of viable cone photoreceptors in the K42E mutants.Peanut agglutinin (PNA) binds to the disaccharide Gal-β(1-3)-GalNAc in glycoproteins and glycolipids . Oligosaan cells . Furtheran cells . Hence, K42E Dhdds mutation specifically associated with RP59 [DhddsK42E/K42E mice, particularly in mice homozygous for the K42E mutation. This expectation was also predicated on a preliminary report [DhddsK42E/K42E mice up to one year of age. Furthermore, despite the confirmed mutation of Dhdds, we found no evidence for defective protein N-glycosylation in the retinas of these mice. The retinas were labeled robustly with fluor-tagged ConA lectin, irrespective of genotype. These findings are in good agreement with observations made by Sabry et al. [N-linked oligosaccharides using either siRNA silencing of DHDDS in a HepG2 cell line or in RP59 (severe mutation) patient fibroblasts. The ConA binding observed in our study is further consistent with observations made by Wen et al., who observed that rather than any loss in dolichols, there was an alteration in dolichol chain lengths (increased D17:D18 ratio) in RP59 patients compared to normal human subjects, but without obvious hypoglycosylation of serum transferrin [Here, we have presented the generation and initial characterization of a novel mouse model of RP59, where we have achieved global homozygous knock-in of the ith RP59 ,4,5. Basith RP59 ,4,5, as ith RP59 ,12,13,14y report , using ay et al.  who founnsferrin . These fDHDDS expression by injection of morpholino oligonucleotides at the one-cell embryo stage [Dhdds knock-down model, the murine RP59 model generated in the present study exhibits robust PNA staining in the outer retina, suggesting persistence of viable cone photoreceptors. In a parallel study , we have observed Dhdds transcript distribution in all retinal nuclear layers by in situ hybridization, consistent with the fact that all cells require dolichol derivatives to support protein N-glycosylation. Taken together, these findings suggest that the K42E Dhdds mutation does not affect cone photoreceptor viability. Recently , we also generated a conditional Dhdds knockout mouse model, with targeted ablation of Dhdds in retinal rod photoreceptors, using a Cre-lox approach; however, unlike the K42E knock-in model, the rod-specific Dhdds knockout model exhibits profound, rapid retinal degeneration, with almost complete loss of photoreceptors by PN 6 weeks. Yet, there was no evidence of compromised protein N-glycosylation prior to the onset of photoreceptor degeneration. In addition, as reported in a companion article in this Special Issue of Cells [Dhdds in retinal pigment epithelium, (RPE) cells in mice also results in a progressive, but somewhat slower, retinal degeneration.Understanding the pathophysiological and biochemical mechanisms underlying RP59 remains limited due to the lack of a validated vertebrate animal model that faithfully mimics the key hallmarks of the disease. Heretofore, only a zebrafish model of RP59 has been documented, using global knock-down of yo stage . In thatof Cells , targeteNus1 gene) [N-glycosylation [DhddsK42E/K42E mouse line and retinal cell type-specific conditional DHDDS knockout mice offer potentially valuable model systems in which to pursue further investigations along these lines. In addition, we are currently pursuing studies employing dual, targeted ablation of DHDDS and NgBR in the retina.  ,9, with sylation ,29. At pse model .)Our findings bring into question the current concept that RP59 is a member of a large and diverse class of diseases known as “congenital disorders of glycosylation” (CDGs) ,31. Whil


Astragalus varieties prescribed by the Chinese Pharmacopoeia, Astragalus mongholicus has better tolerance to UV-B. Thus, it is necessary to study the metabolic strategy of Astragalus under UV-B radiation further. In the present study, we used untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-mass spectrometry (LC-MS techniques) to investigate the profiles of primary and secondary metabolic. The profiles revealed the metabolic response of Astragalus to UV-B radiation. We then used real-time polymerase chain reaction (RT-PCR) to obtain the transcription level of relevant genes under UV-B radiation , which annotated the responsive mechanism of phenolic metabolism in roots. Our results indicated that supplemental UV-B radiation induced a stronger shift from carbon assimilation to carbon accumulation. The flux through the phenylpropanoids pathway increased due to the mobilization of carbon reserves. The response of metabolism was observed to be significantly tissue-specific upon the UV-B radiation treatment. Among phenolic compounds, C6C1 carbon compounds (phenolic acids in leaves) and C6C3C6 carbon compounds (flavones in leaves and isoflavones in roots) increased at the expense of C6C3 carbon compounds. Verification experiments show that the response of phenolics in roots to UV-B is activated by upregulation of relevant genes rather than phenylalanine. Overall, this study reveals the tissues-specific alteration and mechanism of primary and secondary metabolic strategy in response to UV-B radiation.Ultraviolet-B (UV-B) radiation (280–320 nm) may induce photobiological stress in plants, activate the plant defense system, and induce changes of metabolites. In our previous work, we found that between the two  Astragalus mongholicus (A. mongholicus) is an important perennial herb of the Legumes family [O-β-d-glucoside (CAG) can potentially be used as a “marker compound” for the chemical evaluation or product standardization of Radix astragali [s family ,2. Its ds family ,4. Radixs family ,6,7. Calstragali ,9,10.A. mongholicus to capture sunlight for photosynthesis inevitably exposes the plant to ultraviolet (UV) radiation. Ultraviolet radiation is the general term for three radiation wavelengths: UV-C (200–280 nm), UV-B (280–320 nm), and UV-A (320–400 nm). Ultraviolet-C is completely absorbed by atmospheric gases. Ultraviolet-Ais barely absorbed by atmospheric ozone, but UV-A impact on plants is small. Ultraviolet-B is potentially harmful to plants but is partially absorbed by ozone [A. mongholicus and even altered accumulation of bioactive compounds. Given this fact, it is essential to better understand the adaptive responses of metabolites in A. mongholicus to increased UV-B radiation.The need for by ozone ,12,13. Iby ozone . Increasby ozone . The sesby ozone . These sp-coumaric acid, and ferulic acid; and (3) compounds having a C6C3C6 carbon skeleton  [Astragalus mongholicus is considered a rich source of natural phenolic compounds [Phenolic compounds play diverse roles in plants. These compounds provide structural support of the cell wall or protect plants against pathogens, herbivores, and UV radiation . Of all mpounds) ,22. Phenmpounds) ,24. Phenmpounds) . Phenylampounds) . Many inmpounds) . Astragaompounds ,29.Regarding the complex primary and secondary metabolites fluctuations of higher plants, metabolomics plays a vital role as a suitable tool ,30. MetaPAL, E.C. 4.3.1.25) and chalcone synthase , are activated by UV-B [O-glucoside (CAG) biosynthesis pathway has been completely elucidated based on its presence in other legumes [O-glucoside is synthesized from L-phenylalanine via the isoflavonoid branch of phenylpropanoid metabolism. PAL, cinnamate 4-hydroxylase  and 4-coumarate-coenzyme A ligase  are enzymes involved in the upstream general phenylalanine pathway. Isoliquiritigenin, an isoflavonoid skeleton, is synthesized via CHS and chalcone reductase (CHR), co-catalyzed by the condensation of 4-coumaroyl-CoA and three molecules of malonyl-CoA. Afterward, a series of chemical reactions is performed under the catalysis of chalcone isomerase , isoflavone synthase , isoflavone O-methyltransferase (IOMT) and isoflavone 3′-hydroxylase (I3′H). At the last step, the formation of CAG from calycosin (CA) is catalyzed by uridine diphosphate glucose (UDP)-glucose: calycosin 7-O-glucosyltransferase (UCGT). For the last step, the formation of CAG from CAis catalyzed by uridine diphosphate glucose (UDP)-glucose: calycosin 7-O-glucosyltransferase (UCGT) [Many of the biosynthetic enzymes of phenolics, such as phenylalanine ammonia lyase  ,55.A. mongholicus [A. mongholicus to UV-B radiation. To the best of our knowledge, no information on the metabolites response mechanism of A. mongholicus to UV-B radiation is currently available. To deeply explore the response and mechanism explaining how metabolic reprogramming can achieve a new steady state under increased UV-B radiation, we investigated the influence on the specific tissue accumulation of primary and phenolic metabolites using untargeted (GC-MS) and targeted (LC-MS) metabolomics in A. mongholicus under increased UV-B radiation.Due to the release of anthropogenic pollutants such as chlorofluorocarbons, a larger proportion of the UV-B spectrum reaches the surface of the earth and affects all living organisms ,57. In ggholicus ,58. ThisA. mongholicus were sown in Botanical Garden of the Key Laboratory of Plant Ecology, Northeast Forestry University, Harbin, northeastern of China . All plants grew under standard field conditions, with an average temperature of 16 °C during the day (14 h) and 4 °C at night (10 h), and water was applied every two days. After three months, plants of uniform height and stem thickness were transferred to containers (37 cm × 28 cm × 10 cm) and used for this study. All containers (n = 6) contained equal bulk density of potting mix and 12 plants, half of the containers for UV-B treated plants and the other for controlled plants. Plants were treated in the field with UV irradiation . The UV-B irradiation intensity (33.5 μW/cm2) was monitored by a UV light meter . That is, the irradiation doses of UV-B treated group was 804 J m−2, the dose chosen was based on our previous study [2O2 and antioxidant enzymes was performed based on the methods of Liu et al. [Seeds of us study . Controlu et al. ,60,61. Cu et al. ,63.N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (including 1% trimethyl-cholorosilane), and 20 μL were added to the mixture, vortexing for 2 min, which were then derivatized for 60 min in 70 °C. After pretreatment, the solution was centrifuged , after which the supernatant was transferred to a glass vial for GC-MS analysis.Plant samples were pulverized to 5 μm using a grinding instrument , and 90 mg aliquots of roots, stems, leaves, and petioles were extracted. A mixed solution of 40 μL 2-chloro-L-phenylalanine  in 360 μL methanol (pre-cooled at −20 °C) was prepared for metabolite extraction. Metabolites were extracted with the mixed solution by sonication for 30 min, after which they were sonicated again with a chloroform and water solution. Subsequently, the solution was centrifuged at 14,000 rpm (revolutions per minute). The supernatant was collected in a derivatized glass bottle, evaporated to dryness. A derivatization method oximation reaction was then performed to increase the volatility of the metabolites [−1 to 125 °C, 4 °C·min−1 increments to 210 °C, 5 °C·min−1 to 270 °C, and 10 °C·min−1 to 305 °C; and a final holding temperature of 305 °C for 3 min. Mass spectra acquisition parameters were as follows: ion source temperature, 260 °C; filament bias, −70 V; mass range, m/z of 50–600; and acquisition rate, 20 spectra·sec−1 [The GC-MS analysis was performed using a 7890A-5975C system . An Agilent HP-5 (5%-phenyl) -methyl polysiloxane non-polar column was used for GC-MS. The GC process was as follows: an initial temperature of 60 °C; oven temperature increments of 8 °C·minPlant samples were pulverized to 5 μm using a grinding instrument , and 500 mg aliquots of roots, stems, leaves, and petioles were extracted. Samples were extracted with 80% ethanol in water (10 mL) containing 0.1 mg/L lidocaine  by sonication twice, each for 45 min. The sample extractions were filtered, and the filtrates were merged. The filtrates were then dried under low pressure using a vacuum cavitation instrument. The resultant extracted material was dissolved in the mobile phase (1 mL) and filtered through 0.22 μm-diameter micropores. The purified solution was analyzed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS).−1 using the following solvent system: 0.05% acetic acid-water (A), 0.05% acetic acid-acetonitrile (B); 5% B-95% B from 0–23 min; 95% B-5% B from 23–25 min; and 5% B from 25–31 min. Analyses were performed using a UPLC/Q-TOF-MS system (Waters). The MS conditions were set as follows: positive ion mode, capillary voltage of 3.0 kV, cone voltage of 45 V, source temperature of 400 °C, desolvation temperature of 500 °C, cone gas flow of 50 L/h, and desolvation gas flow of 800 L/h. Detection was performed in positive ion mode in the m/z range of 50–1000.Separation was performed on an Acquity UPLC BEH C18 column  with a VanGuard precolumn  and maintained at 30 °C. The volume injected was 2 μL. Gradient elution was performed at a flow rate of 0.25 mL·mint-test combinatory approaches were used to screen for important metabolites (p < 0.05). The LC-MS data were analyzed using the software MassLynx version 4.1. This software detected peaks and listed the detected and matched peaks with the retention time and m/z pair and their corresponding intensities. The relative signal intensities of compounds were standardized by dividing them by the intensities of internal standard. The relationships between 15 primary metabolites and phenolic compounds were used for hierarchical clustering analysis (HCA) by R (www.r-project.org/) for both species. Pearson’s correlation coefficients were calculated for these metabolites, and the Tukey test was performed using Statistical Product and Service Solutions (SPSS) version 17.0. Metabolic pathways were analyzed using the Metaboanalyst web portal  and MBRole (http://csbg.cnb.csic.es/mbrole). The pathways of metabolites were analyzed using database sources including the Kyoto Encyclopedia of Genomes and Genomes (KEGG) (http://www.genome.jp/kegg/) to identify the most affected metabolic pathways and facilitate further metabolite interpretation. The metabolites and corresponding pathways were imported into Cytoscape software version 3.1.0 to visualize the network models. A metabolic correlation distribution network was created from the 144 primary metabolite data using the WGCNA package  [The GC-MS raw data were transformed into NetCDF format by data analysis software (Agilent GC-MS 5975) and were later processed using the software R. Each compound was displayed as the peak area normalized to that of the internal standard. For further analysis, the treated R output data were exported to Microsoft Excel. National Institute of Standards and Technology (NIST) 14 library was searched to compare the structures of the compounds with that of the NIST database. Compounds were then identified based on the retention index and mass spectra with already known compounds in the NIST library. Peak detection, retention time alignment, and library matching were performed using the TargetSearch package from Bioconductor  , after wCA, USA) ,66.The extraction and derivatization of RNA from roots, leaves, stems and petioles were performed as described previously by Liu et al. . Amplifi18S RNA.To determine relative fold differences in template abundance for each sample, the cycle threshold (Ct) values for each of the gene-specific primers were normalized to the Ct value for n = 10) was 19.07% for the control group and 20.70% for the UV-B treatment group. In contrast to the control group, the chlorophyll content decreased in the UV-B treated group. The activity of antioxidant enzymes and content of H2O2 increased under UV-B radiation.The effect of UV-B radiation on morphology can be seen in A. mongholicus under control and UV-B treatments  but did not significantly change in aboveground tissues. Regarding acids, including eight organic acids, one inorganic acid and two fatty acids, diverse trends were observed. Phosphonic acid level decreased under UV-B radiation in all organs. Propanedioic acid increased only in roots, and there was no significant difference in aboveground tissue under control or UV-B treatments. Palmitic acid decreased in belowground tissue (roots) but increased in aboveground tissues (stems) under UV-B radiation. Oxalic acid increased in leaves and stems while hexonic acid increased in leaves and petioles under UV-B radiation. Significant increases in alcoholic compounds in belowground tissues (roots) were observed for d-pinitol, whereas significant increases in aboveground tissues (leaves) were observed for erythritol. In addition, the levels of six other compounds significantly differed from those of the control group under UV-B radiation. Among these compounds, α-d-glucopyranoside levels significantly differed only in belowground tissues (roots).To identify the metabolites that contributed to these variances, 27 significantly differential compounds were identified by their variables of importance in the projection (VIP) values >1, and their s < 0.05 B. There The PLS-DA was used to profile these metabolites. In the PLS-DA score plot, separation of the two treatments and their different tissues was obtained. The samples were clustered into separate groups A. A PLS-To understand the effects of increased UV-B radiation on the level of phenolic metabolites, the targeted phenolic compounds content of the collected samples was analyzed via LC-MS. A total of 29 standard reference compounds were used, all provided by Shanghai yuanye Bio-Technology Co. Ltd , with a purity of ≥98%. The targeted compounds in the samples were identified in accordance with the mass spectral information and retention time of the corresponding standard reference compounds. The mass spectral information has been added as A. mongholicus were determined separately. Visualization of the phenolic profile was performed using HCA  derived from the pentose phosphate pathway (PPP) were also observed in the UV-B treatment group, likely reflecting the higher respiratory rate of the PPP pathway in these plants. In addition, PLS-DA plots of the primary metabolites for four tissues under different conditions were generated , the metabolic mechanisms of these compounds in the enhanced UV-B environment need to be further explored. The CAG biosynthesis pathway and relevant genes are visualized in wounding ,82. The A. mongholicus in an enhanced UV-B environment. We found that in response to UV-B the plant cell is “primed” at the level of primary metabolism by a mechanism that involves the reprogramming of metabolism to efficiently divert carbon toward the aromatic amino acid precursors of the phenylpropanoid pathway. A stronger shift from carbon assimilation to carbon accumulation has occurred. Among the accumulation of phenolics, C6C1 carbon compounds (phenolic acids in leaves) and C6C3C6 carbon compounds (flavones and flavonols in leaves and isoflavones in roots) increased at the expense of C6C3 carbon compounds in order to obtain the stronger UV-B absorptive and antioxidant capacity. Compared with the control treatment, the response of phenolics has a significant tissue-specific in the UV-B radiation treatment. Notably, the response of phenolics in roots to UV-B is activated by upregulation of relevant genes rather than phenylalanine.A metabolic profile was revealed using untargeted GC-MS and targeted LC-MS combined techniques to investigate the tissue-specific metabolic mechanism of


Nature Communications 10.1038/s41467-021-20988-w, published online 3 February 2021.Correction to: The original version of this Article contained errors related to phylogroup attribution of lineages ST-117, ST-657, ST-1163, ST-4673, ST-23, ST-88 and ST-2230.E. coli phylogroups 43, and were absent in phylogroups C and G44,45’. The correct version replaces this sentence with ‘Poultry isolates from our dataset 43-45’.The ‘Results’ originally incorrectly read ‘Poultry isolates from our dataset  in the emergent ST-117 lineage’. The correct version replaces this text with ‘In a second analysis, we tested the ability of the model trained on data from phylogroup A, B1 and B2 isolates, to predict infection status  in the emergent ST-117 lineage (phylogroup G)’.The ‘Discussion’ originally incorrectly read ‘In fact, disease isolates were distributed across the phylogeny within all six previously described phylogroups and the ST-117 complex lineage’. The correct version replaces this sentence with ‘In fact, disease isolates were distributed across the phylogeny within all eight previously described phylogroups’.The original versions of Figs. 1 and 3 omitted to mention that lineage ST-117 belongs to phylogroup G.The original version of Fig. 3a omitted labels for phylogroups C and G and incorrectly labelled isolates from phylogroup C as belonging to phylogroup B1.All the errors have been corrected in both the PDF and HTML versions of the Article.In addition, the original version of the Supplementary Data 1


Multidrug resistance (MDR) is the major obstruction in the successful treatment of breast cancer (BCa). The elucidation of molecular events that confer chemoresistance of BCa is of major therapeutic importance. Several studies have elucidated the correlation of TRPS1 and BCa. Here we focused on the role of TRPS1 in acquisition of chemoresistance, and reported a unique role of TRPS1 renders BCa cells resistant to chemotherapeutic drugs via the regulation of BCRP expression. Bioinformation analysis based on publicly available BCa data suggested that TRPS1 overexpression linked to chemoresistance. Mechanistically, TRPS1 regulated BCRP expression and efflux transportation. Overexpression of TRPS1 led to upregulation of BCRP while its inhibition resulted in repression of BCRP. The correlation of TRPS1 and BCRP was further confirmed by immunohistochemistry in 180 BCa samples. MTT assay demonstrated that manipulation of TRPS1 expression affects the chemosensitivity of BCa cells. In total, high expression of TRPS1 confers MDR of BCa which is mediated by BCRP. Our data demonstrated a new insight into mechanisms and strategies to overcome chemoresistance in BCa. To date, breast cancer (BCa) ranks first among female malignant tumor, accounting for 30%, and the second leading cause of cancer mortality in women, accounting for 15% . MultidrMechanisms of drug resistance include mutations of drug targets, alterations in drug metabolism, increased efflux of anticancer drugs, and the activation of survival or inactivation of downstream death signaling pathways –8. The eTRPS1 gene, of which deletions and mutations cause the tricho-rhino-phalangeal syndromes. To date, increased evidence suggested TRPS1 involved in a wide variety of functions among human malignancies including BCa and prostate cancer . The online tool Kaplan-Meier Plotter was also used for survival analysis  (http://r2.amc.nl). GSE114213 was obtained from Cistrome (http://cistrome.org/db/#/) and visualized by UCSC (http://genome.ucsc.edu/index.html).Datasets of GSE45255, GSE25055, GSE12791, and GSE27830 were downloaded from the Gene Expression Omnibus (GEO) database  . The onl2.The BCa cell lines MDA-MB-231 and MCF-7 were purchased from American Type Culture Collection . MDA-MB-231 cells were cultured in Leibovitz's L-15  Medium containing 10% fetal bovine serum. MCF-7 cells were maintained in Dulbecco's modified Eagle's (Gibco) medium containing 0.01 mg/ml bovine insulin and 10% fetal bovine serum. Both were at 37°C in a humidified incubator supplied with 5% COTo create TRPS1 plasmids, sequences for TRPS1  were cloned into a mammalian expression vector, pcDNA3.1 (+). TRPS1-specific shRNA was purchased from Shanghai Gene Pharma Co., Ltd. shRNA oligonucleotide sequences targeted at TRPS1 were as follows: forward,5′-CACCGCTGCAGAACTAAATCATAAGTTCAAGAGACTTATGATTTAGTTCTGCAGCTTTTTTG-3′; reverse,5′-GATCCAAAAAAGCTGCAGAACTAAATCATAAGTCTCTTGAACTTATGATTTAGTTCTGCAGC-3′. MDA-MB-231 cells transfected with vectors are referred as 231-Vec and 231-TRPS1, and MCF-7 cells transfected with shRNA are referred as MCF7-nc and MCF7-TS.TurboFectTM  was used to stably transfect BCa cells.Total RNA was isolated from cultured cells using RNAiso Plus , and the complementary DNA was synthesized using ReverTra Ace qPCR RT Master Mix  both according to the manufacturers' instructions. mRNA was quantified by real-time PCR analysis using UltraSYBR Mixture with ROX  and ΔΔCt method. β-actin was used as reference gene. The following primer pairs were used for real-time PCR analysis:TRPS1 forward primer, 5′-CAAATCTCAGGCCTGAGTGA-3′, reverse primer, 5′-GTGAAGAGCTGATATCCTGCAG-3′;BCRP forward primer, 5′-TGGCTGTCATGGCTTCAGTA-3′, reverse primer, 5′-GCCACGTGATTCTTCCACAA-3′,β-actin forward primer 5′-CTCCATCCTGGCCTCGCTGT-3′, reverse primer 5′-GCTGTCACCTTCACCGTTCC-3′.Cellular protein extracts were prepared as described previously . The memThe primary antibodies were as follows: goat polyclonal antibody TRPS1 , mouse anti-BCRP (BXP21)  and mouse mAb beta-actin  as a total protein internal control.3 cells per well for 24 h and then incubated with different concentrations of doxorubicin, cisplatin, paclitaxel, 5-Fu , and mitoxantrone  for 48 h. The MTT assay was performed following the manufacturer's instructions. Absorbance was measured using a spectrometric absorbance of 570 nm against a background of 630 nm on a Bio-Rad microplate reader . The dose-dependent curve and 50% inhibitory concentration (IC50) were calculated to determine the chemosensitivity of the cells.The 3-(4.5-dimethylthiazed-2-yl)-2,5-diphenylterazolium bromide  assay was used to assess the chemosensitivity of selected transfection cells to anticancer drugs. The cells were plated in a 96-well plate at a density of 8 × 106 cells were incubated with 25 μM mitoxantrone for 30 min and then allowed to efflux for 2 h in complete medium alone. The cells were washed twice with ice-cold PBS and then resuspended in 500 μl of serum-free medium. All cells were analyzed by flow cytometry  with a 635-nm red diode laser and 670-nm band-pass filter. The fluorescence intensity of control group was set as background to exclude non-specific fluorescence. Data were analyzed with FlowJo 7.6.5 .Mitoxantrone, a known high-affinity substrate of BCRP, was used to detect BCRP transport activity. Approximately 1 × 10Paraffin-embedded samples from 180 patients with invasive ductal BCa were analyzed at the Department of Pathology, Qilu Hospital of Shandong University from 2007 to 2009. Patient consent and approval from the Research Ethics Committee of Shandong University School of Medicine were obtained.Immunohistochemistry was performed as described in previously . The sliThe stained slides were evaluated independently by two independent pathologists (S.P and H.J.) based on previously described semi-quantitatively scoring system. The TRPS1 expression was scored by the product of intensity  and percentage of extent of reactivity . The following cut-off levels were applied: 0 for negative, 1–4 for weak positive, and 6–12 for strong positive . BCRP exp < 0.05 considered as statistically significant. Correlation significance was assessed with Pearson Chi-square test or Fisher's exact test if appropriate.Statistical analysis was performed using SPSS 22.0  and Graph Pad Prism 7 . All data were presented as mean ± SEM, with p = 0.02) and overall survival (p = 0.03), respectively. To further investigate the association of TRPS1 expression and chemoresistance, TRPS1 mRNA expression was analyzed using publicly accessible gene expression data containing chemosensitive and chemoresistant samples. As shown in p < 0.0001), and chemoresistant BCa cells (p = 0.015).Several studies have demonstrated patients with high expression of TRPS1 displayed a lower rate of metastasis. To explore the relationship of TRPS1 expression and prognosis in BCa patients after chemotherapy, Kaplan–Meier plotting and log-rank test were carried out with publicly accessible datasets. Of note, patients after chemotherapy with higher TRPS1 expression exhibited worse distant metastatic overall survival (n = 40103) and BCa dataset (n = 155). TRPS1 expression showed positive correlation to BCRP expression . In the meantime, the relative expressions of BCRP mRNA in MCF7-TS cells were 4.1-fold lower than that of the control cells (p < 0.01). Results of western blot analysis for BCRP proteins showed the same trend as that of qRT-PCR analysis (p = 0.012). These results were further confirmed by Spearman correlation analysis . It has been documented that miR-181a regulated BCRP expression via binding to the 3′-UTR of BCRP mRNA , and multidrug resistance associated protein (MRP1). Although TRPS1 has been associated with P-gp expression in osteosarcoma , the expanalysis . To furtCRP mRNA . BioinfoCRP mRNA  and enriCRP mRNA .The functional efflux assay was based on the extrusion of mitoxantrone, high-affinity substrate of BCRP. Compared to 231-Vec cell, 231-TRPS1 cells exhibited rapid reduction in intracellular mitoxantrone . We alsoWe overexpressed TRPS1 in MDA-MB-231 cells to determine whether TRPS1 affects chemoresistance in BCa cells. Overexpression of TRPS1 in MDA-MB-231 cells (231-Vec/231-TRPS1) was verified by western blot . The selInhibition of TRPS1 in MCF-7 cells (MCF7-ns/MCF7-TS) was verified by western blot . The selThe rapid development of chemoresistance is the major obstacle in the effective treatment of BCa. One of the mechanisms of MDR is the elevated expression of ABC transporters. The ABC transporters increase transmembrane efflux of drugs from cells that causes the decrease of their concentration in cytoplasm and, in consequence of that, insensitivity of cell to damaging impact of cytostatics . BCRP isBCRP, also known as the ATP-binding cassette (ABC) transporter G2 (ABCG2) , 27, is Previous studies have found that hormone nuclear receptors are important determinants of BCRP expression. It has been found that peroxisome proliferator-activated receptor γ (PPARγ) directly regulates the transcription of ABCG2 through binding to the PPAR response elements (PPARE) in the promoter . In BCa,Previous studies have revealed the controversial role of TRPS1 in BCa progression. TRPS1 may cooperate with different co-transcriptional regulators to function as a transcriptional repressor or activator with regard to cell type, microenvironment, stages of development, or pathological conditions . TRPS1 bIn conclusion, we have demonstrated that the expression of TRPS1 is correlated significantly with chemoresistance in BCa. Mechanically TRPS1 promoted BCRP gene expression and efflux transportation in BCa cell sublines. Our observations offer new perspectives for chemotherapy. Despite overexpression of TRPS1 usually predicts a better prognosis in BCa, chemoresistance may develop in patients with high TRPS1 expression. Further investigation involving multicenter clinical samples is warranted.Publicly available datasets were analyzed in this study, these can be found in the NCBI Gene Expression Omnibus .The studies involving human participants were reviewed and approved by Research Ethics Committee of Shandong University School of Medicine. The patients/participants provided their written informed consent to participate in this study.PS and GZ contributed to the conception and design of the study. JH, HZ, and LL performed the experiments presented in the study. Data analysis was carried out by JH and BH. JH and PS were involved in writing the manuscript. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.