Under the reaction temperature of 180 °C and an acetylene gas hourly space velocity (GHSV-C2H2) of 30 h–1, the 15%Sn(II)/PN@AC-550 showed the initial acetylene conversion of 100% and vinyl chloride selectivity over 98.5%. Additionally, the deactivation rate of 15%Sn(II)/PN@AC-550 reached 0.47% h–1, which was lower than that of 15%Sn(II)/AC-550 (1.02% h–1), suggesting that PN@AC-550 as novel support can retarded the deactivation of Sn(II)/AC-550 catalysts during acetylene hydrochlorination. Based on the catalytic tests and characterization results , it demonstrated that PN@AC-550 as support could effectively improve the dispersion of tin species, retard the formation of coke deposition, lessen the oxidation of SnCl2 during the preparation process, as well as relatively inhibit the leach of tin species during the reaction. By combing the FTIR results and Rideal–Eley mechanism, we proposed that that HSnCl3 was transition state of SnCl2 in catalysis acetylene hydrochlorination and then adsorbed the acetylene to produce the vinyl chloride.In this study, the novel tin-based catalysts (Sn(II)/PN@AC) were prepared using the phosphorus and nitrogen dual-modified activated carbon as support and SnCl Despite the initial activity of tin-based catalysts is close to traditional mercury-based acetylene hydrochlorination catalysts, there is still an ongoing study in prolonging the lifetime. The tin-based acetylene hydrochlorination catalysts can be classified into three categories: bimetallic or multimetallic tin-based catalysts [2–8], organotin-based catalysts [9–11], and tin complex-based catalysts . Given the easily sublimation of SnCl4 under industrially relevant process conditions [2], the efficient durability of SnCl4/AC in acetylene hydrochlorination can be prolonged by the incorporation of BiCl3 and CoCl2 [8]. Moreover, the Kocheshkov redistribution reaction of SnCl4 and Ph3ClSn can further improve the catalytic behavior of SnCl4/AC [10]. Later on, Guo et al. founded that the synergistic effect of SnCl2, ZnCl2, and Tb4O7 can strengthen stability of SnCl2/AC in acetylene hydrochlorination [5]. In the previous work, we founded that SnCl2/AC is easily oxidized during preparation process, but Li-Sn(IV) in LiSnCln/AC is structurally stable, enhancing the stability of SnCl2/AC in acetylene hydrochlorination [3]. Moreover, based on the Rideal–Eley mechanism and the previous study and mentioned above results, we can infer that HSnCl3 is a transition state of tin active sites and then adsorbed the acetylene to produce the vinyl chloride [3]. Additionally, the lifetime of SnCl2/AC catalysts in acetylene hydrochlorination can be also elevated via the coordination interaction of organic linkage and Sn sites . Tin-based catalysts for diverse application includes electrochemical CO2/AC in acetylene hydrochlorination [13–15]. Also, nitrogen-doped in Cu-based catalysts for acetylene hydrochlorination can improve the acetylene adsorption and prolong the lifetime [16]. Combing the above results, it is demonstrated that dopants of nitrogen or phosphorus elements into Cu-based catalysts for acetylene hydrochlorination can strengthen the stability [13–18].Owing to the dispersion of Cu species on catalysts surface keeps the direct relation with P doping, Cu-P/AC catalysts displays longer lifetime as compare to CuCl2/AC catalysts for acetylene hydrochlorination. In this work, Sn(II)/PN@AC was prepared using the low-cost phosphoric acid, melamine, and SnCl2 as phosphorus, nitrogen, and tin source, respectively. However, until now few studies researched the effect of nitrogen and phosphate elements on the catalytic performance of SnCl2H4N4, ≥99.5%) and methyl orange was obtained from Tianjin Guangfu Technology Development Co., Ltd. Additionally, phosphoric acid (H3PO4 ≥98.0%) wea purchased from Sinopharm Chemical Reagent Co., Ltd. SnCl2·2H2O (≥98.0%) was bought from Energy Chemical Co., Ltd. Moreover, Na2CO3 (≥99.8%) was obtained from Tianjin Kermel Technology Development Co., Ltd. All materials were used without further purification.Coal-based carbon carrier  was bought from Shanxi Xinhua Chemical Company. Melamine (C3PO4 (0.54 g), C2H4N4 (0.46 g), and AC (9.0 g) were firstly premixed in distilled water at 55 °C for 90 min, after which the sample was dried at 80 °C overnight. The obtained samples were calcinated at 550 °C for 4 h and then denoted as PN@AC. N@AC was prepared by the above-mentioned procedure.H2·2H2O (0.47 g) was completely dissolved in ethanol. Subsequently, SnCl2 solution was gradually added to carbon supports, followed by air drying at 80 °C for 12 h. The above-mentioned described procedures were repeated to prepare 10%Sn(II)/PN@AC, 10%Sn(II)/N@AC, and 10%Sn(II)/AC, respectively.Catalysts were prepared by the following processes. Firstly, SnCl2H2 (VC2H2/VHCl = 1.0:1.1) was introduced into reactor containing 4.0 mL of catalysts with C2H2-GHSV = 30 h–1 or 60 h–1 at 180 °C [19]. The final products contained the unreacted hydrogen chloride, which was adsorbed by the medical soda-lime. Having experienced adsorption, the cleaned gas mixture was analyzed online by GC900 (GDX-301 column).The catalytic performance was tested in a fixed-bed quartz reactor (i.d.=10 mm). To remove water vapor, the reaction system was washed by hydrogen chloride for 30 min before the initial reaction. Then the gas mixture of HCl and CThe BET surface area and pore textural properties data of the catalysts was acquired by a nitrogen adsorption method using Quantachrome Nova2000e instruments. The X-ray diffraction (XRD) patterns of the catalysts was collected from a Shimadzu XRD-6000 instrument using Cu Ka radiation over the range of 10–80 °. Raman spectra of sample were performed on a Renishaw (514 nm laser source). Transmission electron microscopy (TEM) images were obtained using a JEM-2100F instrument with 300 keV acceleration voltages. X-ray photo-electron spectroscopy (XPS) analysis was conducted on an Esca Lab 250Xi spectrometer. Thermogravimetric analysis (TGA) experiments (NETZSCH STA 449F3 Jupiter instrument) carried out to study the coke deposition of catalysts. Inductively coupled plasma optical emission spectrometer (Agilent 720 ICP-OES) was used to determine the absolute content of tin elements in samples. 2H2-TPD) and hydrogen temperature-programmed reduction experiments (H2-TPR) were performed on FINESORB-3010 instruments, respectively [3]. Adsorption capacity of catalysts for HCl were calculated by the titration method [4]. The Fourier transform infrared spectra (FT-IR) of the samples was measured by Biorad Excalibur FTS 3000). Acetylene temperature-programmed desorption  was higher than that of N@AC  and PN@AC . It is demonstrated that the nonmetal additives are filled into the partial pores of AC . Figure 1b shows that the two diffraction peaks of AC were not affected by the introduction of nonmetal additives [20], inferring the well dispersion of promoter on AC surface [21]. As shown in Figure 1c, the ID/IG value gradually declines in the order of PN@AC(1.63)> N@AC(1.23)> AC (0.85), suggesting that PN-doping does lead to the more abundant defect of PN@AC and consequently improve the catalytic behavior of AC in acetylene hydrochlorination [22–24].The structure of different supports was firstly studied, including the BET surface area and the pore volume. The value of AC  is not observed in tin-based catalysts , inferring the dispersion of tin compounds on support surface . As shown in Figures 2b and 2c, numerous black particles are dispersed on the support surface. Meanwhile, Figure 2d illustrates that no large particles are reunited on the PN@AC, indicating that Sn species are stably and homogeneously dispersed on the PN@AC. As evident from Table 1 and Table 2, the load of SnCl2 content (5 wt%–15 wt%). When the SnCl2 content is 15 wt%, Sn(II)/PN@AC achieves an initial acetylene conversion of 100%, with vinyl chloride selectivity of 98.5% . It is noted that the acetylene conversion on 20%Sn(II)/PN@AC does not increase but decrease to 96.8%. However, Sn(II)/AC only behaved about 95.5% acetylene conversion . At same time, the VCM selectivity of Sn(II)/PN@AC is over 98.0%. The results indicated that Sn(II)/PN@AC exhibited highly selectivity and catalytic activity in acetylene hydrochlorination. After 40 h reaction , the acetylene conversion of 15%Sn(II)/AC is 62.1%, while the acetylene conversion of 15%Sn(II)/N@AC decreases from 97.3% to 72.2%, indicating that synergy between Sn species and N is one reason for the longer lifetime. Notably, 15%Sn(II)/PN@AC has an unusual catalytic stability in acetylene hydrochlorination reaction compared to 15%Sn(II)/AC catalysts with the same SnCl2 loading, which indicates that the dual-elements (N and P) additives can further strengthen the stability of 15%Sn(II)/AC . For 15%Sn(II)/N@AC and 15%Sn(II)/AC, the acetylene conversion under C2H2-GHSV of 60 h–1 was 82.7% and 78.5%, respectively, while for 15%Sn(II)/PN@AC, the acetylene conversion under the same reaction condition (60 h–1) was 99.7% . Overall, 15%Sn(II)/PN@AC behaves the acetylene conversion of 99.0%, which meets the industrial production need (C2H2-GHSV=30 h–1 -50 h–1 and T = 130 oC -180 oC ) [25].The catalytic performances of different catalysts were tested in acetylene hydrochlorination, and the results are shown in Figures 3a-c. The acetylene conversion on the Sn(II)/PN@AC increased as the SnCl2+ (~486.7 eV) and Sn4+(~487.7 eV) [26–28]. This result is consistent with H2-TPR results (Table 4). As shown in Figure 4e and Table 4, the H2 reduction peaks of Sn2+ and Sn4+ in catalysts (15%Sn(II)/N@AC and 15%Sn(II)/PN@AC) was lower than the standard consumption peaks (467.4 and 542.9 °C) [26–28], inferring that the nonmetal element additives can retard the oxidation of Sn2+ during the synthesis process and lessen the leach of Sn species during the reaction. The determination of Sn2+ and Sn4+ values in Tables (Table 4 and Table 5) was calculated on the normalization of peak areas. Figure 4c and Figure 4d confirm the existence of N-P and Pyridinic N, which can promote the reactivity of catalysts significantly . The Pyridinic N content of 15%Sn(II)/N@AC was calculated to be 0.9 wt%, which is close to the 15%Sn(II)/PN@AC (0.88 wt%). It is revealed that phosphorus atoms bonded with nitrogen in the pyridine structure (ortho-position NP) is the main factor on the performance of 15%Sn(II)/AC according to the present work  and a recently published paper [25]. The status of tin, nitrogen and phosphorus species in catalysts was characterized by XPS analysis . As Figure 4b illustrates, Sn-based catalysts behaved the two types of Sn species including Sn2 into catalysts shift the P-N binding energy from 133.5 eV to 133.2 eV. Moreover, a positive shift (0.3 eV) can be observed in the binding energy of Sn2+ for 15%Sn(II)/PN@AC versus 15%Sn(II)/AC. Therefore, it is attributing this shift to the synergistic effect between ortho-position NP and Sn species .As shown in Figure 4c, the electron binding energies at 135.0 eV, 133.5 eV, and 132.6 eV can be assigned to P-O, P-N, and P-C . Particularly, the addition of SnCl2, we used FT-IR techniques to analyze three samples including Sn(II)/AC-C2H2, Sn(II)/AC-HCl, and Sn(II)/AC-N2 , which represent that SnCl2/AC was respectively pretreated at 200 °C for 1 h under acetylene, hydrogen chloride, and nitrogen atmosphere, respectively. Two characteristic adsorption bands at ~1121 cm–1 and ~3397 cm–1 are observed in Sn(II)/AC-HCl, suggesting that the gaseous HCl reacted firstly with Sn species. Figure 4a shows that Sn(II)/AC catalysts during acetylene hydrochlorination contains SnCl4 and SnCl2, the latter of which is the main formation of Sn species. Based on the analysis of our previous study [3], owing to SnCl4 and HCl are electron-receptor, the adsorption of HCl on the SnCl2 sites forms the HSnCl3 [34–36]. The results of FT-IR spectra are no obvious difference between Sn(II)/AC-N2 and Sn(II)/AC-C2H2, implying that SnCl2 cannot make bond with C2H2. Combining the Eley–Rideal mechanism and previous work [34–38], it is indicated that HSnCl3 is as transition state of SnCl2 in the catalysis of acetylene hydrochlorination.To study the effect of various adsorption reactants on the structural conformation of SnCl–1, 0.49 mmol·g–1, and 0.28 mmol·g–1, respectively. The published papers finds that HCl adsorbs on Pyridinic N firstly [30], but the Pyridinic N content in 15%Sn(II)/PN@AC is close to 15%Sn(II)/N@AC (Table 6). It is implying that the synergy effect between Sn and NP may be responsible for the highly HCl adsorption capacity and thus improve the catalytic behaviors of tin-based catalysts in acetylene hydrochlorination, based on the above results.HCl adsorption experiments was applied to study the HCl adsorption capacity of catalysts, and the results are displayed in Figure 6a. The adsorption capacity of HCl on 15%Sn(II)/PN@AC, 15%Sn(II)/N@AC, and 15%Sn(II)/AC are 0.64 mmol·g2H2 of catalysts decreases in the order 15%Sn(II)/PN@AC>15%Sn(II)/N@AC>15%Sn(II)/AC. When N and P-codoped 15%Sn(II)/AC can result in higher C2H2 adsorption capacity as compare to 15%Sn(II)/N@AC and 15%Sn(II)/AC. This result indicates that NP is responsible for the C2H2 adsorption capacity, which is well agree with the previous study [25].As shown in Figure 6b, the adsorption area for C2·g–1, 241.1 m2·g–1, and 299.7 m2·g–1. Figure 7 shows the TGA curves of catalysts before and after reaction. Based on the same type fresh- and used-catalysts, the weight loss in the temperature range of 150–470 °C was mainly originated from the coke deposition on the catalysts surface in acetylene hydrochlorination , and the results is listed in Table 7. The amount of coke deposition for the 15%Sn(II)/PN@AC, 15%Sn(II)/N@AC, and 15%Sn(II)/AC catalysts are 1.70%, 3.61%, and 3.70% . Thus, the synergistic effect of Sn species and NP in 15%Sn(II)/PN@AC can accelerate the anti-coking ability, which, consequently, prolonging the lifetime of the Sn-based catalysts. The 75.9%, 72.3%, and 65.4% of the initial Sn content is lost from 15%Sn(II)/AC, 15%Sn(II)/N@AC, and 15%Sn(II)/PN@AC, respectively, after 40 h reaction (Table 8), demonstrating that the leach of tin species is a deactivation reason of tin-based catalysts and manifesting that NP additives can retard the loss of tin compounds during acetylene hydrochlorination[41]. After 40 h reaction, the reduction of BET surface area in used tin-based catalysts is listed in Table 2, the loss of BET surface area in 15%Sn(II)/PN@AC, 15%Sn(II)/N@AC and 15%Sn(II)/AC reaches 138.6 m2 as catalyst exhibits considerable catalytic performance, its toxicity and sublimation can be led to the serious environmental pollution. Additionally, the Minamata convention will be forbidden the utilization of mercury-based materials. But this study reported that Sn/PNAC as catalysts for acetylene hydrochlorination was prepared using nontoxic compounds. This work also finds that the stability of 15%Sn(II)/PN@AC catalysts correlate with N and P additives. After careful characterizations and additional catalytic tests, PN@AC supports can not only make the tin compounds dispersion well, but also strengthens the reactants adsorption of catalysts. According to the XPS, C2H2-TPD, H2-TPR, and HCl adsorption experiments results, it is found that the better catalytic behavior of Sn(II)/PN@AC is mainly attributed to the synergy between ortho-position NP and Sn species. Additionally, the reaction mechanism was proposed as follows, the adsorption of HCl on SnCl2 forms the transition state (HSnCl3) is the initial step and then react with C2H2 to produce the vinyl chloride. Such a finding provides the guidance to develop the tin-based catalysts for acetylene hydrochlorination. Although carbon supported-HgCl


Chemotherapy remains the most common cancer treatment. Although chemotherapeutic drugs induce tumor cell senescence, they are often associated with post-therapy tumor recurrence by inducing the senescence-associated secretory phenotype (SASP). Therefore, it is important to identify effective strategies to induce tumor cell senescence without triggering SASP. In this study, we used the small molecule inhibitors, UNC0642 (G9a inhibitor) and UNC1999 (EZH2 inhibitor) alone or in combination, to inhibit H3K9 and H3K27 methylation in different cancer cells. Dual inhibition of H3K9me2 and H3K27me3 in highly metastatic tumor cells had a stronger pro-senescence effect than either inhibitor alone and did not trigger SASP in tumor cells. Dual inhibition of H3K9me2 and H3K27me3 suppressed the formation of cytosolic chromatin fragments, which inhibited the cGAS-STING-SASP pathway. Collectively, these data suggested that dual inhibition of H3K9 and H3K27 methylation induced senescence of highly metastatic tumor cells without triggering SASP by inhibiting the cGAS-STING-SASP pathway, providing a new mechanism for the epigenetics-based therapy targeting H3K9 and H3K27 methylation. In recent years, several strategies for the treatment of cancer have been developed; however, chemotherapy remains the preferred strategy for the treatment of multiple tumors. Tumor cell senescence induced by chemotherapeutic drugs is as an important cytological mechanism for the inhibition of tumor . HoweverCytoplasmic chromatin fragments (CCF) are associated with cellular senescence ,10,11. IAccumulating evidence suggests that epigenetic mechanisms, such as histone modification, are associated with the pathogenesis of multiple cancers; therefore, targeting histone modification maybe a promising therapeutic strategy for cancer . MethylaIn the present study, we revealed a novel mechanism underlying tumor cell senescence following epigenetics-based therapy whereby dual inhibition of H3K9me2 and H3K27me3 promoted tumor cell senescence without triggering secretion of SASP. These results supported the use of histone methyltransferase inhibitors as a promising epigenetics-based therapy.H3K9 and H3K27 methylation inhibits gene transcription and plays essential roles in the pathogenesis of multiple cancers ,30,31,32Initial experiments  showed aOur initial experiments showed that dual inhibition of H3K9me2 and H3K27me3 promotes tumor cell senescence . HoweverWe confirmed that dual inhibition of H3K9me2 and H3K27me3 induced tumor cell senescence without triggering SASP, which differed from the effect of doxorubicin . HoweverAlthough CCF promote SASP in senescent cells , they doIn this study, we found that dual inhibition of H3K9me2 and H3K27me3 using a combination of UNC0642 and UNC1999 exerted a stronger pro-senescence effect in malignant cancer cells than single inhibition. Dual inhibition of H3K9me2 and H3K27me3 suppressed SASP by decreasing the formation of CCF through a mechanism involving cGAS-STING signaling .Epigenetic mechanisms such as histone modification are associated with human diseases, especially cancer . The metCell senescence is a relatively stable state of cell cycle arrest and an important cytological mechanism for the prevention of cancer . Tumor cChemotherapy can increase intratumoral heterogeneity, leading to different fates of tumor cells: senescence or apoptosis . Many gev/v) horse serum, 20 ng/mL EGF, 0.5 mg/mL hydro cortisone, 100 ng/mL cholera toxin, 10 mg/mL insulin, and 10 U/mL penicillin-streptomycin. MDA-MB-231 cells were cultured in L-15 medium  with 10% (v/v) fetal bovine serum . HEK293T, MCF7, BT549, SW480, HCT116, PC3, and DU145 cells were cultured in DMEM (Sigma-Aldrich) with 10% (v/v) fetal bovine serum . All cell lines were grown at 37 °C with 5% CO2 except MDA-MB-231, which was cultured at 37 °C without CO2.Cell lines  were obtained from the American Type Culture Collection . These cells were characterized by DNA fingerprinting and isozyme detection and were tested with a MycoBlue Mycoplasma Detector  to exclude mycoplasma contamination before the experiments. MCF10A cells were cultured in DMEM-F12 supplemented with 5% (UNC1999 was purchased from MCE (MedChemExpress HY-15646). UNC0642 was purchased from MCE . Doxorubicin (Dox) was obtained from Selleck (S1208). Cell lines were treated with a single inhibitor (UNC1999 or UNC0642) or a combination of the two inhibitors (UNC1999 + UNC0642) or with DMSO for up to seven days  or nine days , refreshing the medium and drugs every three days.The pCDH-CMV-Flag-dnMCAK expression vector , and theReverse transcription, PCR, and real-time PCR were performed, as described previously . The priWestern blotting was performed, as described previously . AntibodImmunofluorescence was performed, as described previously .Wound healing was performed, as described previously .Cell cycle assay was performed, as described previously .SA-β-gal staining was performed, as described previously .3 cells/100 μL/well) were plated in triplicate in 96-well plates and incubated with fresh medium for 24 h. Then, the cells were incubated with a single inhibitor (UNC1999 or UNC0642) or a combination of the two inhibitors (UNC1999 + UNC0642) or with DMSO for up to seven days, refreshing the medium and drugs every three days. Cell viability was assessed on days one, three, five, and seven using the Cell Counting Kit-8 (CCK8)  or MTT Assay Kit  and a microplate reader , according to the manufacturer’s instructions.To assess the antiproliferative effects of the EZH2 and G9a inhibitors, tumor cell lines  was used to calculate the significance of differences between groups. Data were considered significant at p < 0.05 . Statistical analysis was performed using GraphPad Prism software .The results were compiled from at last three independent replicate experiments and are presented as the mean ± SD. The unpaired Student’s In conclusion, we demonstrated that the combination of an EZH2 inhibitor and a G9a inhibitor induced senescence in highly metastatic tumor cells by targeting H3K9me2 and H3K27me3 and without triggering SASP, providing a potentially promising epigenetics-based therapeutic strategy for the treatment of highly metastatic tumors. Moreover, a combination of EZH2 inhibitor, G9a inhibitor, and other chemotherapy drugs may be an effective therapeutic strategy for the treatment of tumors in the future.


Escherichia coli JM109. The fusion protein containing a GST tag was purified, and New Zealand white rabbits were immunized to produce the HPV16 E7 polyclonal antibody. The anti-HPV16 E7 antibody was verified by western blotting and immunofluorescence stains, and applied in 182 HPV16 DNA-positive cervical tissue specimens and matched 36 HPV DNA-negative specimens by immunohistochemistry. Furthermore, A positive correlation between HPV16 E7 protein expression and malignancy grade was observed. But there is no relationship between HPV16 E7 protein expression and tumor sizes, tumor differentiation, lymph node metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage, or lymphovascular space invasion in cervical cancer. These findings provide a basis for further research focusing on the role of HPV E7 protein in various HPV-related diseases.Almost all cervical cancer is associated with persistent infection of high-risk (hr) human papillomavirus (HPV) like HPV16. The E7 oncoprotein encoded by hrHPV plays a crucial role in carcinogenesis. The present study aimed to establish a reliable protocol of immunohistochemistry stains to detect HPV16 E7 protein in formalin-fixed and paraffin-embedded tissue specimens of various cervical lesions. Firstly, the HPV16 E7 gene was inserted into a prokaryotic expression vector pGEX-4T2. Then the recombinant plasmid pGEX-4T2-(HPV16-E7) was transformed into  Cervical cancer is the fourth most common cancer and the fourth leading cause of cancer death in women worldwide. According to the World Health Organization's global cancer observatory, there were an estimated 570,000 new cases and 311,000 deaths in 2018 worldwide. Besides, the incidence rate was 2-fold higher, and the mortality rate was 3-fold higher in low/medium Human Development Index (HDI) regions compared with high/very-high HDI regions The HPV genome consists of approximately 7,900 base pairs that can be divided into three regions: an early (E) region, a late (L) region, and a non-coding long control region containing regulatory elements for viral DNA replication and transcription. The L genes encode structural proteins. The E region contains six open reading frames  that are mainly for regulatory functions engaged in genome persistence in vitro, preparation of polyclonal anti-HPV16 E7 antibody, and its clinical application in various cervical lesions to investigate the relationship between HPV16 E7 protein expression and multiply clinical features.HPV genotyping analysis was widely used in clinical practice and provided great prevention against invasive cervical carcinomas This study was approved by the Ethics Committee of the Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University and conducted with written informed consents of all participants. Formalin-fixed and paraffin-embedded cervical tissue specimens and their corresponding patient data were obtained from the Departments of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University between 2009 and 2016 by cervical biopsy or hysterectomy. Patient data including tumor size, tumor differentiation grade, lymphovascular invasion, and lymph node metastasis was reviewed respectively. All patients were staged according to the International Federation of Gynecology and Obstetrics stage system, histologically classified and graded according to the WHO classification criteria, cytologically classified according to the Bethesda classification by two experienced pathologists. The inclusion criteria were described as follows: (1) polymerase chain reaction (PCR) proved HPV16 DNA-positive cervical lesions, (2) pathologists get consensus with histological classification, grade and cytological classification. The exclusion criteria were described as follows: (1) patients received chemotherapy, radiotherapy or immunotherapy before specimen collection. (2) tumor recurrence.All animal procedures were performed in accordance with animal use protocols approved by Zhejiang Chinese Medical University Animal Care and Use Committee and Zhejiang University Animal Care and Use Committee. New Zealand white rabbits  were supplied by the Laboratory Animal Research Center of Zhejiang Chinese Medical University  to raise polyclonal antibodies. Human embryonic kidney (HEK) 293T cell line and human cervical squamous cell carcinoma SiHa cell line were cultured in Dulbeccos modified Eagles Medium (DMEM) supplemented with 4.5g/L glucose and 10% fetal bovine serum .BamHI and EcoRI restriction enzyme sites were synthesized by Qingke Biological Technology Co., Ltd. . The PCR product was cleaved with BamHI and EcoRI, purified and ligated to the BamHI- and EcoRI-digested pGEX-4T2 vector. pGEX-4T2-(HPV16-E7) encodes the HPV16 E7 protein containing a GST open reading frame. The Escherichia coli strain JM109  was used as the host. The resultant plasmid was further confirmed to be correct by double restriction enzyme digestion and sequencing .The HPV16 E7 gene was amplified by PCR from HPV16 genomic DNA . The forward primer (5′ CCGGGATCCATGCATGGAGATACACCTAC 3′) and the reverse primer (5′ CCGGAATTCTTATGGTTTCTGAGAACAGATG 3′) containing The strains harboring HPV16 E7 were grown in LB medium containing 100μg/ml ampicillin shaken at 250 rpm and 37°C until the optical density (OD) 600nm reached 0.6-0.8. Isopropyl β-D thiogalactopyranoside with a final concentration of 0.2mM was added, and the culture was incubated at 26°C for 4-6 hours. Cells were harvested by centrifugation, resuspended in PBS, and sonicated on ice. The homogenate was centrifuged at 2,000 × g for 5min, both supernatants and pellet fractions were collected. The fusion protein was purified by affinity chromatography with glutathione-Sepharose 4B beads , the GST-tag was removed by thrombin , the purified protein was dialysed against PBS overnight and stored at -80°C.To produce anti-HPV16 E7 polyclonal antibodies, New Zealand white rabbits were immunized by subcutaneous injection of about 500 μg HPV16 E7 (in 0.8ml) mixed with an equal volume of Freund's complete adjuvant . Booster injections were applied as described above but using Freund's incomplete adjuvant at 10-day intervals three times. The quality of the antibodies in sera was monitored by indirect enzyme-linked immunosorbent assay (ELISA), which was repeated daily until a threshold was determined .For ELISA, as the antigen for ELISA, purified HPV16 E7 proteins were diluted to a final concentration of 50 μg/ml in 0.05 M carbonate buffer solution (pH 9.6) at 37°C for one hour and at 4°C overnight. After washing with PBS containing 0.05% Tween 20 and blocking with 200 μL 5% milk at 37℃ for 2 hours, 100 μL anti-HPV16 E7 polyclonal antibody  was used as the primary antibody, and HRP-conjugated goat anti-rabbit antibody was used as the secondary antibody. TMB Solution was used as the color substrate and terminated with TMB Stop Solution, the OD450 was detected. Serums with the OD450 values higher than that of control serums by 2.1-fold were defined as positive.Another 500 μg HPV16 E7 without adjuvant was injected ten days later. Rabbit sera were collected five days after the last immunization. The Protein G Agarose (Life Technology) was applied to purify polyclonal immunoglobulin according to the manufacturer's protocol. The serum was stored at -20°C in aliquots.TM, USA). The rabbit polyclonal antibodies raised against HPV16 E7 were used as the primary antibodies. The peroxidase-conjugated anti-rabbit immunoglobulin G  was used as the secondary antibodies. Protein was detected with Enhanced Chemiluminescence (ECL) western blotting substrate  through a chemiluminescence imaging system .For western blotting, cell lysates and protein samples were subjected to SDS-PAGE and transferred to PVDF membrane . Membranes were blocked in PBS-T containing 5% w/w non-fat milk  and permeabilized with 0.1% Triton X-100 . Anti-HPV16 E7 polyclonal antibody was used as the primary antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG  was used as the secondary antibody. DAPI (Beyotime) was applied to stain the nucleus and stainings were observed under an immunofluorescence microscope .Immunohistochemistry stains were performed as follows. Formalin-fixed paraffin-embedded tissue sections (8 μm) were deparaffinized in xylene and rehydrated in graded ethanol. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0). Slides were treated with 0.3% hydrogen peroxide for 30 minutes to quench endogenous peroxidase activity, blocked with PBS containing 10% normal goat serum at room temperature for 30 minutes. Rabbit anti-HPV16 E7 polyclonal antibody (1:400) was used as the primary antibody and the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000) was applied as the secondary antibody. Slides were then incubated with DAB  for 5 minutes, counterstained with hematoxylin for 5 seconds, visualized using a microscope .Five random high power fields (400×) were observed to score the staining intensities and positive cell percentages. The staining intensities were quantified as follows: 0 (no staining); 1 (light brown); 2 (brown); 3 (dark brown). The positive cell percentages were graded as follows: 0 (0%); 1 (1-25%); 2 (26-50%); 3 (51-75%); 4 (76-100%). The immunoreactive score  was finally obtained by calculating the product of the staining intensity (0-3) and the positive cell percentage (0-4). According to the immunoreactive score, the staining results were defined as follows: negative, score 0-1; low expression, score 2-3; moderate expression, score 4-5; high expression, score ≥ 6. The results were evaluated by two independent pathologists.P value < 0.05 were considered statistically significant.Spearman's rank correlation test was used for comparison between groups. All statistical analyses were performed using the SPSS 20.0 statistical software . BamH I and EcoR I  vectors were identified by gene sequencing and double enzyme digested with The HPV16 E7 polyclonal antibody was obtained from the rabbits that were immunized five times with purified HPV16 E7 protein. Rabbit serum antibody concentrate was monitored by ELISA and purified by the Protein G Agarose. The specificity of the purified anti-HPV16 E7 polyclonal antibody was verified by both western blotting and immunofluorescent stains Figure . SiHa cep < 0.01), HPV16 E7 protein expression levels and histological grade , and HPV16 E7 protein expression positive rates with cytological malignancy grade  (Table p < 0.01). The negative coincidence rate was 100% accordingly. However, for the different tumor sizes, tumor differentiation, lymph node metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage, or lymphovascular space invasion in 58 cases of cervical cancer, there is no relationship between these subgroups in HPV16 E7 protein expression levels .Immunohistochemistry stains of HPV16 E7 protein were applied in a total amount of 182 specimens of PCR proved HPV16 DNA-positive cervical lesions and 36 matched HPV16 DNA-negative chronic cervicitis tissue specimens Figure . It alsoCXCL14 to promote tumor growth via angiogenesis The HPV infection is associated with a broad spectrum of diseases that range from self-limiting skin warts to life-threatening cancers. The hr-HPV has been confirmed with close links to invasive cervical cancer, especially HPV16 and 18. Moreover, E7 protein encoded by hr-HPV types plays a crucial role in carcinogenesis by the following mechanism: Initially, E7 oncoproteins might down-regulate TLR-9, a fundamental role in pathogen recognition and innate immune activation, to evade the immune response and cause persistent infection There are various methods to detect HPV16 E7 protein, from detecting antibodies to HPV16 E7 protein in serum by radioimmunoprecipitation assay Previous studies indicate that both HPV16 E7 mRNA in vitro may provide a basis for further research focusing on the role of HPV E7 proteins in various cancers.In conclusion, the present study demonstrates the preparation of HPV16 E7 polyclonal antibody and a reliable immunohistochemistry protocol for its application in various cervical lesions. The positive coincidence rate of HPV16 E7 protein immunohistochemistry stain compared to HPV genotyping analysis in CIN1, CIN2, CIN3, and cervical cancer was 15%, 93%, 100%, and 100%, while the negative coincidence rate was 100% accordingly. It revealed an elevated HPV16 E7 protein-positive rate in HPV16 DNA-positive cervical lesions with increasing histological and cytological malignancy grades. However, there is no relationship between HPV16 E7 protein expression levels and tumor sizes, tumor differentiation, lymph node metastasis, FIGO stage or lymphovascular space invasion in cervical cancer. The preparation of the HPV16 E7 protein


Aberrant Wnt/β-catenin signaling contributes to the development, progression, and metastasis of CRC, by altering target gene expression connected to cancer cell proliferation and motility. S100A4 is a Wnt/β-catenin target gene, which strongly enhances migration and invasion of CRC cells and thus CRC metastasis. Here, we report the transcriptional cross-regulation of S100A4 and the Wnt antagonist DKK1, in which the expression of S100A4 down-regulates DKK1 expression, sustaining activated Wnt signaling. S100A4 is an established prognostic biomarker for CRC patient survival, and the combination of S100A4 and DKK1 can be used to improve the prognosis of overall and metastasis-free survival.Metastasis is directly linked to colorectal cancer (CRC) patient survival. Wnt signaling through β-catenin plays a key role. Metastasis-inducing S100A4 is a Wnt/β-catenin target gene and a prognostic biomarker for CRC and other cancer types. We aimed to identify S100A4-dependent expression alterations to better understand CRC progression and metastasis for improved patient survival. S100A4-induced transcriptome arrays, confirmatory studies in isogenic CRC cell lines with defined β-catenin genotypes, and functional metastasis studies were performed. S100A4-regulated transcriptome examination revealed the transcriptional cross-regulation of metastasis-inducing S100A4 with Wnt pathway antagonist Dickkopf-1 (DKK1). S100A4 overexpression down-regulated DKK1, S100A4 knock-down increased DKK1. Recombinant DKK1 reduced S100A4 expression and S100A4-mediated cell migration. In xenografted mice, systemic S100A4-shRNA application increased intratumoral DKK1. The inverse correlation of S100A4 and DKK1 was confirmed in five independent publicly available CRC expression datasets. Combinatorial analysis of S100A4 and DKK1 in two additional independent CRC patient cohorts improved prognosis of overall and metastasis-free survival. The newly discovered transcriptional cross-regulation of Wnt target S100A4 and Wnt antagonist DKK1 is predominated by an S100A4-induced Wnt signaling feedback loop, increasing cell motility and metastasis risk. S100A4 and DKK1 combination improves the identification of CRC patients at high risk. Adenomatous Polyposis Coli (APC) are found in 70–80% of patients [Colorectal cancer (CRC) is a major cause of cancer death worldwide and particularly in Western countries . Turningpatients . APC trupatients . The majpatients .2+ binding protein S100A4 [One of the Wnt signaling target genes in CRC is the metastasis-inducing small Can S100A4 ,12. Its n S100A4 ,14. Thern S100A4 ,16,17 ann S100A4 ,19,20,21Wnt/β-catenin signaling also regulates the expression of Dickkopf-1 (DKK1) . DKK1 itIn this study, we aimed to explore S100A4-induced transcriptome alterations in CRC cells. We discovered a so far undescribed feedback loop within the Wnt pathway. S100A4, the expression of which is induced by active Wnt/β-catenin signaling, suppresses the expression of DKK1. We analyzed this transcriptional cross-regulation of metastasis-inducing S100A4 and the Wnt antagonist DKK1 in CRC in cell culture and confirmed it in CRC xenografted mice. In addition, we found the activating transcription factor 5 (ATF5) involved in the expression regulation of DKK1 in CRC cells with restored low Wnt/β-catenin pathway activity. Combinatorial analysis of inverse S100A4 and DKK1 expression in human CRC patient specimens improved prognostication for patient survival.The CRC cell line HCT116 (heterozygous gain-of-function Δ45-β-catenin), and the single allele derivatives HAB68 (Δ45-β-catenin) and HAB92 (wild-type β-catenin), were kindly provided by Todd Waldman . Plasmid transfection was performed with FugeneHD , according to the manufacturer’s instructions. Transfection of HAB92 and HCT116 cells with pcDNA3.1 or pcDNA3.1/S100A4 resulted in the control cell lines HAB92/vector and HCT116/vector, and in the S100A4 overexpressing cell lines HAB92/S100A4 and HCT116/S100A4, respectively. Transfection of HCT116 cells with S100A4-specific (shS100A4) or unspecific control shRNA-plasmids (shCtrl) resulted in HCT116/shS100A4 and HCT116/shCtrl cells, respectively. HAB92/shDKK1 and HAB92/shCtrl cells were generated by transfecting DKK1-specific (shDKK1) or unspecific (shCtrl) shRNA-plasmids . Generated cell lines with modulated target gene expression were generated from clonal expansion of selected stably resistant cells after transfection.2, 95% humidity). Lyophilized recombinant human DKK1 (rDKK1) protein  was dissolved in PBS, supplemented with 1% BSA . Cells were treated for 24 h with the indicated rDKK1 protein concentrations. Control cells were treated with the same amounts of 1% BSA solution alone.Cell lines SW620, LS174T, Colo205, SW480, HCT15, LoVo, HT29, Caco 2, WiDr, KM12, SW48, and DLD-1 were obtained from ATCC. Authentication of the cell lines was verified by short tandem repeat (STR) genotyping at the DSMZ . All cell lines were tested regularly for the absence of mycoplasma. The cell lines were cultured in recommended culture medium, supplemented with 10% FBS . Cells were grown in sterile conditions in a humidified incubator  (accessed on 30 April 2011) and online tools from the ‘Database for Annotation, Visualization and Integrated Discovery’ (DAVID) [The competitive hybridization cDNA microarrays were performed at the Center for Cancer Research/NCI . Each experiment consisted of two identical microarrays containing reciprocally labeled cDNAs from test samples (HAB92/S100A4) and control samples , giving 4 arrays for analysis of expression differences. The test sample was stained with Cy5 (red fluorescence) and the control sample with Cy3 (green fluorescence). In reciprocal arrays, test samples were stained with Cy3 and controls were stained with Cy5. In brief, isolated total RNA from HAB92, HAB92/vector, and HAB92/S100A4 cells was reverse transcribed and labeled with either Cy5 or Cy3 dye. Test samples were combined with their respective control samples and hybridized onto Human OncoChip arrays (NCI). Fluorescence intensities were determined with a GenePix 4100A microarray scanner. The data were analyzed by GenePix Pro 4.1 software, and the microarray intensities were normalized by setting the ratio of medians to 1. Data were analyzed with available tools on mAdB ( (DAVID) ,35.Total RNA from cultured cells was extracted with Trizol RNA extraction reagent (Life Technologies). Total RNA from micro-dissected tumor tissues was isolated using the Universal RNA Purification Kit . RNA was reverse transcribed as described previously . cDNA quWestern blot analysis was performed as previously described . Briefly5 cells were plated into 6-well plates, and the cell-free supernatant was harvested after 24 h. The supernatant was diluted with blocking reagent  before entering the previously blocked wells. rDKK1, dissolved in blocking reagent, was used as a standard. Each experiment was performed in duplicate with at least two different dilutions to assure that ELISA reaction occurred in the linear range of sensitivity. The mean values of secreted DKK1 from each supernatant were normalized to the amount of total protein extracted from the respective cells.Secreted DKK1 was quantified with the DuoSet human DKK-1 ELISA System (R&D Systems). In brief, 4 × 106 cells were plated in 15 cm dishes 24 h prior to performing the assay. Cells were incubated with 13.5% formaldehyde for 10 min at room temperature to assure reversible cross-linking of proteins, washed twice with ice-cold PBS, and lysed according to the manufacturer’s instructions . Cell lysates were sonicated for 20 pulses at 100% output and centrifuged at 10,000 rpm at 4 °C for 10 min. The upernatant was transferred to a new tube and aliquoted in 50 μL aliquots for incubation with antibodies. A total of 5 μL of supernatant was stored at −20 °C and used as an input control. Each ChIP was incubated with 10 μg antibody or 10 μg control IgG overnight at 4 °C. Non-bound proteins were washed, followed by elution of the protein-DNA complex from the beads according to the manufacturer’s instructions. Cross-linking of protein and DNA was reversed, and PCR amplification of the DKK1-promoter was performed with a limit of 35 cycles, using the following primer set: pDKK1-fow: 5′-cgactaagcaagggagggg-3′; pDKK1-rev: 5′-gcctttataccgcgggcc-3′. PCR product was analyzed via 2% agarose gel electrophoresis.A total of 1 × 104 cells in a 24-well plate and directly transfected with 500 ng reporter plasmid using TransIT-2020  according to the manufacturer’s instructions. After 48 h, cells were transfected with a mix of 100 ng DKK1-promoter plasmids (firefly luciferase) and 20 ng renilla luciferase plasmid (Promega) as an internal control. Cells were lysed 24 h after transfection, and reporter assay was performed using the Dual Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. Firefly and Renilla luciferase activities were measured using an infiniteM200Pro  plate reader.For transient transfection, cells were seeded at a density of 4 × 105 cells were seeded into each transwell chamber, treated with 100 ng/mL rDKK1 or control solution, and incubated for 24 h to migrate through the membrane. After insert removal, cells at the bottom chamber were trypsinized and counted in a Neubauer chamber . Each well was counted ten times. The experiments were performed in duplicates and repeated twice.Filter membranes of 12 μm pore size (Millipore) were used to analyze the migratory ability of HCT116/vector and HCT116/S100A4 cells. A total of 2.5 × 10Animal experiments were performed in accordance with the United Kingdom Co-ordinated Committee on Cancer Research (UKCCCR) guidelines and were approved by the responsible local authorities , with the registration number A0010/19. mRNA samples of intrasplenically xenografted CRC mouse tumors were obtained after systemic treatment with shRNA expression plasmids. Experimental procedures were previously described . In brie2O2/PBS. Cells were permeabilized with 0.5% Triton-X-100/PBS and unspecific binding sites blocked with 1% horse serum/PBS. Target-specific antibodies  were applied overnight in 0.1% horse serum/PBS and detected with a rabbit-specific DAB kit  according to the manufacturer’s instructions. Nuclei were stained with hematoxylin, and pictures were taken with a BZ-X800 microscope  at 20× and 40× magnification. Quantification of IHC signal intensities was performed with ImageJ (v1.53).Cryosections of the tumor tissue (spleen) were fixed with 4% paraformaldehyde, quenched with 0.1 M glycine/PBS, and residual cellular peroxidase activity was blocked with 0.9% Hwww.ncbi.nlm.nih.gov/geo) (accessed on 10 December 2018). Expression data of target genes were normalized to G6PDH and analyzed for direct or inverse correlation. Expression data of the following sets were combined after normalizing: GDS2201 [Publicly available expression data of CRC tumor microarrays were obtained from Gene Expression Omnibus , with the registration number AA3/03/45. Primary tumor tissues were collected immediately after surgical removal and snap-frozen in liquid nitrogen according to internal protocols. In addition to routine pathological examination of the tumor tissue, the histopathology of each sample used for experimental analysis was reviewed by an experienced pathologist to confirm diagnosis, tissue composition, and tumor content. Tumor staging and typing were performed according to UICC and WHO guidelines. The patients were preoperatively untreated, had no history of familial CRC, did not suffer from a second tumor, and underwent R0 resection. One cohort consists of tumor samples from 41 CRC patients at stages I–IV. For the second cohort, CRC tumor tissues were obtained from 60 CRC patients at stages I, II, or III, i.e., without distant metastases at the time point of diagnosis. Detailed information on patients and tumor tissue of both cohorts are provided in previous reports . All tumt-test was used to compare two groups of data. Comparison of more than two groups was performed by one-way analysis of variance (ANOVA) and Bonferroni post hoc multiple comparisons, or one-way ANOVA on ranks and Tukey post hoc multiple comparison, if the normality test of the data failed. Pearson correlation analysis was used to identify expression correlations. Survival rates were calculated with Kaplan–Meier estimator, with multiple comparisons (pairwise over strata) if indicated. The cut-offs to distinguish low and high expression levels were determined using Receiver–operator-characteristics (ROC) analysis by taking the value with the highest Youden-Index. p-values less than 0.05 were defined as statistically significant. All computations were performed using IBM SPSS Statistics 21.Student’s n ≥ 3).Although many protein–protein interaction partners of S100A4 have been identified that sustain the pro-metastatic action of S100A4 , much leThe results of four microarrays were combined and subsequently analyzed. A total of 324 transcripts were found differentially expressed in dependency of S100A4 overexpression, with 195 transcripts showing a more than four-fold difference. A total of 32 of the functionally annotated genes in this set were up-regulated, and 140 were down-regulated a. When wp = 0.003; p < 0.001; p < 0.001; p < 0.001; p < 0.001; p < 0.001; One of the most highly and consistently down-regulated genes in the arrays was DKK1 a. This rp = 0.041). The differences in mRNA expression levels were verified at the protein level by either WB for S100A4 , on the basis of ChIPseq data from Lovo cells (GSM1208713) [p = 0.026; p = 0.014; By determining the S100A4-induced transcriptome in CRC cells, we were interested in which transcription factors are involved in the respective expression regulation of differentially expressed transcripts, focusing on the 140 down-regulated genes a. An in 1208713) . We obsep = 0.009; p = 0.022; p = 0.015) and an increase in S100A4 protein levels in HAB92/shDKK1  less S100A4 mRNA, whereas treatment with 100 ng/mL rDKK1 reduced the S100A4 mRNA level to 68%  or a non-targeting control shRNA (HCT116/shCtrl). HCT116/shS100A4 cells express 56% less S100A4 mRNA, compared to the control cells HCT116 and HCT116/shCtrl, which was confirmed at the protein level . We fouThe reciprocal expression regulation of S100A4 and DKK1 in vivo was further validated by a lower abundance of human DKK1 in mouse plasma when ectopic S100A4 expression was induced in transplanted HAB92 cells .n = 224). The inverse correlation of S100A4 and DKK1 mRNA expression in CRC patient tumors was confirmed by Pearson correlation analysis  . For S10With the above-reported transcriptional cross-regulation of S100A4 and DKK1 expression in mind, we combined the survival analyses of S100A4 and DKK1 expression. The correlation for patients’ OS and their expression levels of the respective genes increased the significance when based on a combinatorial analysis of S100A4 and DKK1. If patients expressed low levels of S100A4 and high levels of DKK1 in the tumor, the five-year survival was 90% (±7.0%) in the first cohort c and 91%Taken together, the combination of S100A4 and DKK1 expression enables more powerful prognostication of patients’ outcomes. Patients with high S100A4 and low DKK1 expression levels in the primary tumor can be classified as high risk for OS. For MFS, patients with low intratumoral S100A4 expression become high-risk patients when the DKK1 expression is also decreased.Here we report the transcriptional cross-regulation of the Wnt target genes S100A4 and DKK1 by exploring the first S100A4-regulated transcriptome in CRC. Knock-down of S100A4 under constitutive active Wnt signaling restored the expression of DKK1 in vitro and in vivo. As overexpression of S100A4 reduced DKK1 mRNA and protein levels, S100A4 can be seen as the predominant factor in this feedback loop in Wnt signaling modulation. The inverse correlation of S100A4 and DKK1 was validated in publicly available CRC expression datasets. Combining the intratumoral expression levels of S100A4 and DKK1 increased OS and MFS prognostication and identification of CRC patients at high risk.S100A4 expression is a marker for malignancy in several cancer types, including CRC ,47. SomeA recent publication by Park et al. describes remaining susceptibility to Wnt signaling pathway regulation by Wnt stimulation or APC regulation even in the presence of an S45Δ-β-catenin gain-of-function mutation, such as in HCT116 cells . The proWe found ATF5, a member of the ATF/cAMP response element-binding protein family, involved in the regulation of DKK1 expression in CRC cell lines, depending on the activity of the Wnt/β-catenin signaling pathway. ATF5 itself has been related to cell enhanced invasion of fibrosarcoma and breast cancer cells , and itsWhile constitutively active Wnt signaling in the colon gives rise to adenocarcinoma, elevated levels of S100A4 in the primary tumor drives cancer progression up to the formation of distant metastases ,13. S100The diagnosis of metastasized CRC is correlated with the worst prognosis for CRC patients . With thTherapeutic approaches to restore the expression of DKK1 in tumors, with subsequent reduction of up-regulated Wnt target genes, combined with a reduction of S100A4 expression, could improve the outcome of S100A4-driven CRC. In recent reports, DKK1 expression in CRC cells was restored by treatment with Genistein or targeting the vitamin D receptor, leading to reduced Wnt target gene expression ,67. FurtTaken together, the identification of S100A4-mediated transcriptome revealed the transcriptional cross-regulation of the metastasis-inducing S100A4 and the Wnt antagonist DKK1, dominated by S100A4, which leads to increased cell motility, cancer progression and metastasis, and decreased survival of CRC patients. By combining both genes in expression analyses of CRC tumors, we were able to identify high-risk patients who might benefit from adapted cancer therapy.


Escherichia coli (NMEC) is the second leading cause of sepsis and meningitis in neonates worldwide. Here, we report the genome sequence of NMEC15, belonging to serotype O18:K1, isolated from the cerebrospinal fluid (CSF) of an infant with neonatal bacterial meningitis (NBM) in the Netherlands.Neonatal meningitis  Escherichia coli (NMEC) is the second leading cause of sepsis and meningitis in neonates worldwide  was isolated from the cerebrospinal fluid (CSF) of a newborn infant (<28 days) with meningitis in the Netherlands as described elsewhere , 7. The https://github.com/tseemann/shovill) with the SPAdes v3.15.3 assembler and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.2 .NMEC15 was grown on LB agar and subsequently in Luria-Bertani broth at 37°C. Genomic DNA (gDNA) was extracted using the ChargeSwitch gDNA mini bacteria kit  for Illumina sequencing. The DNA yields were quantified using a Qubit fluorimeter double-stranded DNA (dsDNA) high-sensitivity (HS) kit (Life Technologies). The QIAseq FX kit  was used to prepare the genomic library for Illumina 2 × 300-bp MiSeq sequencing. The raw reads were subjected to quality processing using Trimmomatic v0.40 to remove low-quality reads/regions and Illumina adapters. Unless otherwise stated, all software used default parameters. The reads were then assembled using Shovill (GPLV3) (AP) v5.2 . FollowiTwo clusters for the type 6 secretion system (T6SS) were identified in NMEC15, T6SS1 and T6SS2, using NCBI BLAST analysis of contigs. The T6SS1 cluster is 30.2 kb long, with a GC content of 52.2%. The T6SS2 cluster is 27.9 kb long, with a 52% GC content.sul1 (sulfonamide) and aadA1 (aminoglycoside). The E. coli virulence factors identified through VirulenceFinder v2.0 included the increased serum survival gene (iss), the S-fimbria minor subunit (sfaS), and the vacuolating autotransporter toxin (vat).The resistance genes identified using ResFinder v4.1 included SRX10988923 (NMEC15), BioProject accession number PRJNA732675, BioSample accession number SAMN19334708, and genome accession number JAHTGQ000000000.The genome sequence has been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number


Cancer cells develop resistance to chemotherapeutic intervention by excessive formation of stress granules (SGs), which are modulated by an oncogenic protein G3BP2. Selective control of G3BP2/SG signaling is a potential means to treat non-small cell lung cancer (NSCLC).Co-immunoprecipitation was conducted to identify the interaction of MG53 and G3BP2. Immunohistochemistry and live cell imaging were performed to visualize the subcellular expression or co-localization. We used shRNA to knock-down the expression MG53 or G3BP2 to test the cell migration and colony formation. The expression level of MG53 and G3BP2 in human NSCLC tissues was tested by western blot analysis. The ATO-induced oxidative stress model was used to examine the effect of rhMG53 on SG formation. Moue NSCLC allograft experiments were performed on wild type and transgenic mice with either knockout of MG53, or overexpression of MG53. Human NSCLC xenograft model in mice was used to evaluate the effect of MG53 overexpression on tumorigenesis.mg53-/- mice. Transgenic mice with sustained elevation of MG53 in the bloodstream demonstrate reduced tumor growth following allograft transplantation of mouse NSCLC cells. Biochemical assay reveals physical interaction between G3BP2 and MG53 through the TRIM domain of MG53. Knockdown of MG53 enhances proliferation and migration of NSCLC cells, whereas reduced tumorigenicity is seen in NSCLC cells with knockdown of G3BP2 expression. The recombinant human MG53 (rhMG53) protein can enter the NSCLC cells to induce nuclear translation of G3BP2 and block arsenic trioxide-induced SG formation. The anti-proliferative effect of rhMG53 on NSCLC cells was abolished with knockout of G3BP2. rhMG53 can enhance sensitivity of NSCLC cells to undergo cell death upon treatment with cisplatin. Tailored induction of MG53 expression in NSCLC cells suppresses lung cancer growth via reduced SG formation in a xenograft model.We show that MG53, a member of the TRIM protein family (TRIM72), modulates G3BP2 activity to control lung cancer progression. Loss of MG53 results in the progressive development of lung cancer in Overall, these findings support the notion that MG53 functions as a tumor suppressor by targeting G3BP2/SG activity in NSCLCs.The online version contains supplementary material available at 10.1186/s12943-021-01418-3. Lung cancer is a leading cause of mortality worldwide, afflicting approximately 170,000 people each year in the United States, and non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases . In 2020Under stress conditions, such as hypoxia, nutrient deprivation, heat shock, or oxidative stress, cells have to save energy by silencing or reducing protein synthesis for survival. One mechanism to regulate protein synthesis is the formation of stress granules (SGs) –8. TheseA recent series of studies from our group identified MG53  as an important component of cell membrane repair –35. MiceIn addition to facilitating tissue repair, MG53 contains a conserved RING domain with intrinsic E3-ligase activity –44 and cmg53-/-, wild type, and tPA-MG53 mice, and found aggressive lung tumor growth in the mg53-/- mice compared with wild type mice, whereas lung tumor growth was significantly suppressed in the tPA-MG53 mice. Consistent with findings from other investigators  (5 mg/mL in PBS) was added to each well and further incubated for 4 hr. Absorbance was recorded at 570 nm after adding 150 μL DMSO.Primary antibodies used in this study are as follows: anti-PABP-1 and anti-tubulin , anti-Ki67 (Sigma), anti-G3BP2  [TM Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cell and tissue lysates were used for immunoblotting, as previously described [TM Gel Imaging System (Bio-Rad).The cytoplasmic and nuclear protein extraction were performed by NE-PERescribed . BrieflyFor Co-immunoprecipitation (co-IP): HEK293 cells were transfected with HA-MG53 (full length or deletion mutants) and Myc-DDK-tagged G3BP2 (Origene) for 24 hr and incubated in media containing proteasome inhibitor MG132  for 16 hr. Cells were re-suspended in RIPA buffer plus protease inhibitor cocktail (Sigma) and pre-cleared with a mixture of protein-A and protein-G beads (KPL Inc.). Immunoprecipitation was performed with anti-HA magnetic beads (ThermoFisher). The resulting immuno-complexes were collected and separated by SDS-PAGE.Confocal images were taken with Zeiss 780 or Nikon A1R confocal microscope. The images were analyzed using ImageJ software.Histology and immunofluorescent staining were performed as previously described , 39. BriAll data are expressed as means ± SD. For each experiment, three independent replicates were performed. Statistical evaluation was conducted using Student t-tests and by ANOVA for repeated measures (Graphpad Prism 8.2). A value of P<0.05 was considered statistically significant.mg53-/- mice, we noticed an interesting phenomenon: mg53-/- mice often develop lung tumors as they age. As shown in Fig. mg53-/- lung sections with aggressive tumor proliferation, which were positive for staining using the solid tumor marker Ki67 [mg53-/- mice  showed high Ki67-staining in the lung sections, whereas only 11% (2 out of 18) age-matched wild type mice demonstrated such aggressive proliferation pattern.Studying ker Ki67 . ApproxiBrafV600E/+Atg7−/−)50 into wild type, mg53-/-, and tPA-MG53 mice [6 cells were injected into the flanks of mice subcutaneously. At 4 weeks after transplantation, solid tumors were explanted and weighed . We found that MG53 showed various expression levels in the different patient samples (which may reflect the different injury status of the lung tissue), but on average, there were no significant differences between tumor and non-tumor samples (p=0.865) .Through collaboration with the Tissue Repository Center at The Ohio State University, we obtained 20 human NSCLC patient samples from tumor and adjacent non-tumor lung tissues. Western blot showed that G3BP2 protein was up-regulated in tumor (T) versus non-tumor (N) samples  and non-tumor regions . G3BP1 has been widely used as a biomarker of SG [arrow marks). Similar observations were also made in lung sections derived from mg53-/- mice (10 months age), which contained more cytosolic granule structures than those from the wild type mice  staining using antibodies against G3BP1 and G3BP2 with the paired lung sections derived from the tumor  studies demonstrated a physical interaction between MG53 and G3BP2 . Interestingly, we found that cells treated with arsenite trioxide . We constructed RFP-MG53 (human MG53 sequence) and G3BP2-GFP fluorescent constructs, which allowed us to follow the subcellular distribution of MG53 and G3BP2 under live-cell confocal imaging. Clearly, there was significant co-localization between RFP-MG53 and G3BP2-GFP in A549 cells under basal condition  or G3BP2 (shG3BP2). Western blot assay confirmed the reduced expression of MG53 in shMG53 cells Fig. A, and reG53 Fig. C. ColonyG53 Fig. D and E.FTo determine whether knockdown of MG53 and G3BP2 affected cell migration and invasion, we performed a matrigel-based transwell assay. The cell migration capacity was enhanced by ~20% in sh-MG53 cells and reduced by ~60% in shG3BP2 cells, compared with the control cells Fig. F and G.Using MTT assay, we compared the dose-dependent effect of rhMG53 on the proliferation of Scr control and shG3BP2 cells Fig. H and I. Using ATO-induced oxidative stress model , A549 ceG3BP1 and PABP-1 are functional and spatially linked to SGs , 15, 59.In vitro assay with A549 cells was conducted to evaluate the combinatory effect of rhMG53 and cisplatin to cause death of lung cancer cells. We first conducted experiments to establish the dose-dependence of cisplatin to induce death of A549 cells by incubating the cells with varying concentrations of cisplatin for 48 hours or 72 hours  -inducible secretion of MG53 from lung cancer cells Fig. A. A tissright panel) with the concurrent secretion of MG53 into the culture medium was observed.As shown in Fig. in vivo study with transplantation of A549 cells infected with Ad-TRE-tPA-MG53 into the flanks of the immune-deficient NCR nude mice. As shown in Fig. lower left panel). Similarly, IHC staining with antibody against MG53 supported Dox-induced elevation of MG53 in the explanted xenograft; and many cells displayed nuclear-pattern of MG53 localization .We next performed an mg53-/- mice, we found that the loss of MG53 led to the age-dependent development of lung cancer. Using allograft transplantation of mouse NSCLC cells into the mg53-/-, wild type, and tPA-MG53 mice, we demonstrated a physiological role of MG53 in the modulation of lung cancer growth. Knockout of MG53 leads to aggressive lung tumor growth whereas elevated levels of MG53 in circulation inhibit lung cancer tumor growth. We showed that NSCLC tissues derived from human patients contain elevated levels of G3BP2 with excessive formation of SGs. Knockdown of MG53 enhanced proliferation and migration of NSCLC cells, however knockdown of G3BP2 reduced migration and proliferation of NSCLC cells. Furthermore, the exogenous rhMG53 protein had anti-proliferative effect on human NSCLC cells, and such effect was lost with knockout of G3BP2. We conducted in vitro studies with multiple NSCLC cell lines, including A549, H460, and H2122 cells, and found that ATO-induced SGs could be inhibited by treatment with rhMG53. The tumor suppressor function of MG53 was further demonstrated using a xenograft model of lung cancer where inducible MG53 expression were shown to have an inhibitory role on tumor growth.Since the discovery of MG53 in 2009 , many stG3BP2 is an important regulator of the Ras signal pathway with oncogenic function, and also the main component of SGs. The TRIM family proteins are characterized by the conserved tripartite motif, including a RING finger, a B-box, and the coiled-coil domain . Throughin vitro study with cultured NSCLC cells, where rhMG53 treatment led to more nuclear translocation of G3BP2. Our in vitro study with cultured A549 cells support the potential synergy between MG53 and cisplatin to induce death of lung cancer cells. It is conceivable that a combination of rhMG53 with the conventional chemotherapeutic reagents can increase the effectiveness of these drugs to treat late-stage or drug-resistant NSCLCs. More in vivo studies are required to evaluate the potential combinatory effect of rhMG53 and cisplatin to determine their ability to inhibit tumor growth of drug-resistant NSCLCs, or other aggressive cancers with abnormal function of G3BP2/SG.Cancer cells use SG formation to support survival and metastatic capacity when the cells are exposed to adverse environments , 24, 74.We have done biochemical studies to show that MG53 did not control the protein level of G3BP2 in NSCLCs. Yet, MG53 has profound effect in modulating the nuclear translocation of G3BP2 under stress conditions. Many studies in cancer biology also demonstrate that modulation of the cytosol/nuclear compartmentalization of proteins with oncogenic or tumor suppressor functions have significant impact on tumorigenesis . For exaMG53, as a muscle-derived protein that circulates in the bloodstream, is known to facilitate repair of injury to multiple vital organs , 82. TheIn summary, data from the current study characterized a novel function of MG53 in the modulation of G3BP2-mediated SGs associated with lung cancer tumorigenesis. rhMG53 protein can potentially be used as an adjuvant to enhance the therapy of NSCLCs. Since the TRIM domain of MG53 contains the potential active motif(s) to facilitate interaction with G3BP2, targeting the TRIM/G3BP2 interaction can potentially be explored as alternative means to treat lung cancers.Additional file 1: Supplementary Figure S1. Human NSCLC patients show elevated both G3BP1 and G3BP2 expression. The whole protein extracts from the additional 10 human NSCLC tumor were subjected to western blot analysis with anti-G3BP1 and G3BP2 antibodies compared with matching non-tumor lung tissues. Actin served as a loading control. Western blot shows elevated levels of both G3BP1 and G3BP2 in human NSCLC cancer tissues (T) compared to adjacent non-tumor lung tissues (N). The multiple bands of G3BP2 detected in the western blot may reflect the different splice variants of G3BP2 that are present in the human lung tissue.Additional file 2: Supplementary Figure S2. Knockout of MG53 results in elevated stress granule formation in mg53-/- lung. (A) The whole protein extracts from wild type and mg53-/- lung were subjected to western blot analysis with anti-MG53 and G3BP2 antibodies. GAPDH served as a loading control. (B) Representative images of IHC staining of G3BP2 (red) from WT and mg53-/- mice lungs. The nucleus was stained with DAPI (blue). Arrows indicate stress granules.Additional file 3: Supplementary Figure S3. rhMG53 treatment suppresses SG formation and enhances G3BP2 nuclear translocation in multiple NSCLC cells. SGs were induced by ATO treatment. (A) A549 cells were treated with control (top panels), rhMG53 (10 μg/mL) (2nd panels), ATO (0.5 mM) (3rd panels), or ATO (0.5 mM) plus rhMG53 rhMG53 (10 μg/mL) (lower panels) for 40 min, and the cells were analyzed by IHC staining with anti-G3BP1 and anti-G3BP2. G3BP1 was used as a SG marker. The nucleus was stained with DAPI (blue). Arrows indicate G3BP2 nuclear localization. (B) H460 cells were treated with control (top panels), rhMG53 (10 μg/mL) (2nd panels), ATO (0.5 mM) (3rd panels), or ATO (0.5 mM) plus rhMG53 rhMG53 (10 μg/mL) (lower panels) for 40 min, and the cells were analyzed by IHC staining with anti-PABP-1 and anti-G3BP2. PABP-1 was used as SG marker. The nucleus was stained with DAPI (blue). Arrows indicate G3BP2 nuclear localization.Additional file 4: Supplementary Movie. A549 cells were transfected with G3BP2-GFP and MG53-RFP. After 24 hr of transfection, the culture media was changed to fresh media containing Hoechst 33342 and ATO (0.5 mM) was added at the beginning of the live cell imaging. Serial live cell images were taken using Nikon A1R laser microscope . Representative cell images before ATO addition  or at 30 min after ATO treatment were presented in Fig.


Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV  emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions. Influenza is an acute respiratory infectious disease caused by influenza virus infection. Influenza viruses belong to the family Orthomyxoviridae; there are four main types  . InfluenThe smallest nonstructural protein (NS) segment encodes two kinds of viral proteins (NS1 and NS2/NEP). The length of NS1 protein varies from 215–237 amino acids, depending on the strain of virus . TherefoHowever, the NS1 protein of the 2009 pandemic H1N1 IAV (pH1N1) virus lost the ability to bind CPSF30, so it cannot block expression of host genes, although studies show that this ability can be restored by introducing amino acid changes R108K, E125D, and G189D ,12,13. IHere, we sequenced a seasonal 2009 H1N1 influenza virus isolated in 2018 and confirmed the presence of mutations I123V and N205S in important functional sites. However, 108R and 189G were conserved when compared with the early pandemic strain A/California/07/2009. To increase our understanding of the antagonistic mechanisms underlying interactions between influenza virus and host innate immunity, we used a reverse genetics approach to introduce a double mutation (R108K/G189D) into the NS1 protein of the seasonal 2009 H1N1 IAV, rescued the mutant virus, and analyzed its biological characteristics. We found that the mutated NS1 protein harboring R108K/G189D exhibited systematic and selective inhibition of cytokine responses beyond the complexity of host–influenza NS1 protein interactions.2 in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% PSG . IAV A/Urumqi/XJ49/2018 (H1N1) (XJ49) was isolated from throat swabs obtained from patients in Urumqi in 2018 (GenBank Accession no.: MN549338–MN549345).MDCK (Madin–Darby canine kidney) cells , HEK293T (Human embryonic kidney) cells , and A549  cells  were cultured at 37 °C/5% COhttps://www.ncbi.nlm.nih.gov/FLU, accessed on 10 November 2020). We used Molecular Evolutionary Genetic Analysis (MEGA) software v6.0 and Weblogo  for sequence alignment and mutation site analysis.According to their lineage/subtype, as well as the year, 2155 seasonally circulating NS1 sequences were downloaded from the influenza virus database of National Center for Biotechnology Information randomly . The rXJ49 H1N1 virus was generated using an eight-plasmid system based on pHW2000 by transfection into human HEK293T cells. To obtain pHW2000 plasmids containing eight segments of IAV XJ49, viral RNA was extracted from infected cells and cDNA synthesized by reverse transcription using Uni12 primers . The cDNTo access virus growth kinetics in vitro, triplicate wells containing confluent monolayers of MDCK cells  were infected with viruses at a MOI of 0.01. After 1 h of virus adsorption at 37 °C, cells were overlaid with DMEM containing TPCK-treated trypsin (2 μg/mL) and then incubated at 37 °C. At 0, 12, 24, 48, 72, and 96 h post-infection (hpi), 500 μL cell supernatant was collected from each well, and the viral titer was determined in a plaque assay.Confluent monolayers of MDCK cells (105 cells/well in a 12-well plate) were infected with virus at 37 °C. After 1 h of virus adsorption, cells were overlaid with DMEM containing 1% Low Melting Point agarose and TPCK-treated trypsin (2 μg/mL) and then incubated at 37 °C for 3 days. At 3 dpi, cells were fixed for 15 min at room temperature with 4% paraformaldehyde. The agarose overlay was removed and stained for 10 min with 1% crystal violet in methanol. The number of plaques in each well was counted.Samples of transfected cells were prepared in buffer containing 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 20% mercaptoethanol. The samples were heated for 15 min at 100 °C and loaded onto an SDS–PAGE gel. Proteins were transferred to a PVDF membrane, blocked with PBST buffer containing 5% bovine serum albumin (BSA)/0.1% Tween-20, and then probed at room temperature for 2 h with anti-NS1 polyclonal antibody . An anti-GAPDH monoclonal antibody  was used as a loading control. A horseradish peroxidase (HRP) secondary antibody  was used to detect primary antibody binding. Protein bands were visualized using SuperSignal West Femto’s highest sensitivity chemiluminescence substrate kit .50 of influenza virus, four groups of mice (n = 5/group) were anesthetized with pentobarbital sodium (240 mg/kg body weight). One group was used as a negative control group (PBS-infected), another was used as a positive control group (CA07), and the remaining groups were infected with virus. Of these, one group was infected with XJ49-NS1mut virus (XJ49-NS1mut), and one group was infected with rXJ49 virus (rXJ49). The MLD50 of each virus was calculated using the method of Reed and Muench. Survival and body weight of each individual mouse was monitored daily for 15 days. Mice were sacrificed and scored dead when weight loss reached 25% of the baseline weight . Mice were sacrificed at 1, 3, and 6 dpi, and lungs were extracted and homogenized prior to measurement of virus titer. Virus titers were determined in a plaque assay in MDCK cells. Lungs were collected at 6 dpi and fixed with 4% neutral phosphate-buffered formalin. Fixed specimens were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). For immunohistochemical staining, sections were probed with antibodies specific for the A/H1N1 HA protein or mouse IFN-β. Tissue sections were examined under a light microscope ). Cytokine and chemokine expression in lung homogenates was measured using a ProcartaPlex Multiplex Immunoassay kit .Five-week-old female C57BL/6N mice were purchased from Charles River . All animal experiments were approved by the Ethical Committee of Beijing Institute of Microbiology and Epidemiology. To determine the MLDThe 293T cells cultured in 24-well plates were transiently co-transfected with the reporter plasmid p125-luc encoding FF-Luc under the control of the IFN promoter, a plasmid pRL constitutively expressing Rluc under the control of the HSV–TK promoter, and the WT and mutant NS1 pHW2000-expressing plasmids (pHW2000-XJ49NS or pHW2000-NS1mut), using Lipofectamine 3000 reagent . The empty pHW2000 plasmid was used as a negative control, while poly (IC) was used as a positive control. At 24 h post-transfection, cells were harvested and lysed. Reporter activity was analyzed using a Dual-Luciferase Reporter Assay System kit . All assays were performed independently and in triplicate.ΔΔCT method. The results are expressed as the n-fold difference relative to expression of GADPH (2−ΔΔCT).Total RNA was extracted from cultured cells or lung homogenate using a PureLink viral RNA/DNA Mini kit . Real-time PCR was performed using a One Step TB Green PrimeScript PLUS RT-PCR Kit  and a Light Cycler 480 II . The specific primers used to target human interferon and the amplification and quantitative real-time PCR (qRT-PCR) conditions can be provided upon request. Relative expression was calculated using the comparative Culture supernatants were collected from A549 cells and assayed using the human interferon enzyme-linked immunosorbent assay kit .t-test, and differences between three or more groups were evaluated using analysis of variance (ANOVA). Statistical significance was set at p < 0.05. .Data are presented as means ± SD of at least three independent experiments. Differences between two groups were evaluated using Student’s To analyze whether the NS1 protein encoded by the human pH1N1 virus affects the growth and pathogenicity of the virus, the NS gene sequences of 2155 strains of H1N1 virus isolated since 2009 were downloaded and analyzed. Since the influenza A/H1N1 pandemic in 2009, NS1 proteins have evolved different proportions of R108 and K108, as well as of G189 and D189. Collective analysis of all available NS1 sequences of pdmH1N1 indicated that arginine (R) and glycine (G) are the predominant amino acids at positions 108 and 189, respectively A. The re50 of WT rXJ49 was 10−2 while that of rXJ49-NS1mut virus was 10−1.3  influenza viruses in vivo, The results showed that recombinant rXJ49-NS1mut was more pathogenic than WT rXJ49 virus A. The MLs 10−1.3 B. By cons 10−1.3 A,C.Pathological analysis of lung tissue from infected mice was performed at 6 dpi to further examine the pathogenic effects of rXJ49-NS1mut. Compared with those in the mock group  treatment was used as a positive control. After 24 h, the activity of FF–Luc was analyzed. As shown in To clarify how the XJ49-NS1mut virus affects host innate immune responses in vitro, A549 cells were infected with the indicated viruses, and expression of mRNA encoding key innate immune molecules  was measured by qPCR C after 2Mice were infected with the WT rXJ49 virus or the rXJ49-NS1mut virus. At 6 dpi, mice were sacrificed, and lungs were extracted and homogenized. Cytokine and chemokine expression in lung homogenates was measured using the ProcartaPlex Multiplex Immunoassay. When a virus infects the host, it stimulates the immune system, which tries to remove the virus; here, we found that expression of cytokines involved in multiple pathways leading to IFN production were upregulated A; the exThe pathogenesis of IAV depends on the interaction between virus proteins and the host immune system. To replicate successfully in the host, IAVs have evolved to evade host immune response by encoding proteins such as NS1, which regulate host responses.Previous studies show that the IAV NS1 protein binds to CPSF30, which suppresses expression of host genes encoding IFN and ISGs, thereby counteracting innate immune responses . The NS1Here, we compared the sequences of 2155 strains of H1N1 virus identified since 2009 and found that, although some had R108K and G189D mutations in the NS1 protein, most retained R108 and G189. In 2018, we isolated a seasonal pandemic H1N1-origin influenza virus strain called A/Urumqi/XJ49/2018 from throat swabs of patients. We sequenced it and found that the NS1 protein contained the E125D mutation but retained R108 and G189. The isolate exhibited much lower morbidity in a mouse model than the early pandemic 2009 H1N1 isolate CA07. Thus, human H1N1 virus with triple or individual mutations at residues 108, 125, and 189 seemed not to be emerging constantly. Here, we used a H1N1 reverse genetics system to introduce mutations at sites 108 and 189 of the NS1 protein, hoping to obtain information about the molecular mechanism underlying how polymorphisms at these residues affect the interaction between NS1 and host factors. As expected, the rXJ49-NS1mut virus harboring the mutant NS1 protein replicated in MDCK cells in a manner similar to that of WT XJ49 virus but was more pathogenic in C57BL/6N mice. A previous study showed that a triple mutant NS1 virus based on the CA07 backbone showed less morbidity, whereas the mutant NS1 virus based on the XJ49 backbone exhibited higher morbidity. Nogales et al. revealed that inhibition of host gene expression depends on a strict balance between the NS1 and PA-X proteins. Compared with the pandemic CA07 virus, the seasonal XJ49 virus contained N204S, R221Q, L229S mutations in PA-X, which may be the reason for the different morbidity, even though it contained the same triple R108K, E125D, and G189D mutations [Inhibition of interferon-induced cellular signals is very important for influenza infection, because production of cytokines such as type I interferon is the initial innate immune response that fights infection. The NS1 protein, which weakens the IFN response through a variety of mechanisms during influenza virus infection, is the most important IFN-antagonistic protein encoded by the virus . The proThe TRIM family is a superfamily with Ring-finger E3 ubiquitin ligase activity, which has more than 70 members in humans. TRIM family members are critical regulators of innate immune responses against microbial pathogens, including viruses . During It seems that different cytokine waves appear as infection progresses. The first cytokines produced in the lungs after IAV infection are chemokines CCL5/RANTES, CCL2/MCP-1, and CXCL8/IL-8, as well as the main antiviral cytokines IFN-α, IFN-β, and IFN-γ. When the virus reaches the lower respiratory tract, it can infect activated alveolar macrophages and secrete higher levels of proinflammatory and antiviral cytokines . In the


SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 ( IGHV) as patients with a high level of somatic mutations in IGHV (M-CLL) have a better prognosis than patients with no or a low level of somatic mutations in this region (U-CLL) [Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and mainly affects elderly people . Althoug (U-CLL) ,2,3.del(17p) and mutations in tumor protein P53 (TP53). Additionally, shorter progression-free survival is conferred by mutations in the splicing factor 3b subunit 1 (SF3B1), ATM serine/threonine kinase (ATM), ribosomal protein 15 (RPS15), and Notch receptor 1 (NOTCH1) [SF3B1, encoding a component of the splicing machinery. Patients with SF3B1 mutations (SF3B1mut) have a poor prognosis [SF3B1 alterations are associated with chemotherapy refractory disease [SF3B1 mutations cluster in the HEAT repeat domain , protein phosphatase 2A (PP2A), and the yeast kinase TOR1) with the most common mutation in CLL being p.K700E [SF3B1 transcripts by small interfering RNA (siRNA) in cell culture leads to an alteration of exon usage, in most cases causing a decrease in cassette exon inclusion [SF3B1 mutations [Sf3b1+/− mice exhibited a similar phenotype as PcGmut mice, i.e., various skeletal alterations along the anterior-posterior axis [SF3B1mut CLL is still largely missing.Through the development of new high-throughput sequencing technologies, additional genomic alterations have been identified, which are associated with poor prognosis or insufficient therapy response. The strongest impact has been found for (NOTCH1) ,7,8,9,10nclusion . Long reutations . Recentlutations . As suchutations  and withior axis . Furtherior axis . HoweverIGHV (U-CLL) and is associated with a poor prognosis, whereas HP-CLL is enriched for mutated IGHV (M-CLL) [Epigenomic alterations are emerging as powerful prognostic indicators in CLL ,18. It h (M-CLL) .Recent studies have revealed wide-spread intratumor heterogeneity of the methylation in CLL ,26,27. USF3B1, we compared genome-wide methylation profiles of CLL patients with (SF3B1mut) and without (SF3B1WT) SF3B1 mutations.Moreover, all three DNA methyltransferases (DNMTs) are subjected to alternative splicing ,30,31 anSF3B1 mutations on the epigenome is unclear. We used DNA of 27 patients with  and without  SF3B1 mutations   < 0.05, and |log2(fold change)| ≥ 1)  between the methylation levels of the regions estimated with MeDIP-seq/QSEA and EpiTYPER  . Using te)| ≥ 1) . We valie)| ≥ 1)  and obseEpiTYPER . AlthougEpiTYPER . A hiera SF3B1WT A.SF3B1mut effect, did contain methylation data of mutSF3B1 patients. Kulis et al. used 139 CLL patients, 8 of which with SF3B1 mutation [SF3B1mut and SF3B1WT. Although 74 of these 94 CpGs (79%) were within genomic regions sufficiently covered by sequencing reads in our data and tested for differential methylation, none of the DMRs identified overlapped the exact differentially methylated CpGs identified by Kulis et al. [BCL9L, MYB, and NCOR2. The CpGs and DMRs within these genes had the same direction of the methylation change; they were hypomethylated in CLL with SF3B1mut compared to CLL with SF3B1WT. We next used an even larger dataset, one that encompasses the dataset from Kulis et al. [SF3B1 mutational status (148 SF3B1WT and 26 SF3B1mut) were analyzed for 435,102 CpGs . We used the limma R package [SF3B1mut patients with a maximum |log2(fold change)| = 0.3, within the ACOX3 gene (n = 8), or B cell-specific (n = 8) [SF3B1WT and CLL SF3B1mut of the 18 CpGs were significantly correlated between the array and the MeDIP-seq based data    (Table S 0.0038) .SF3B1mut samples are enriched in certain genomic regions or if they are evenly distributed across the genome. While we identified DMRs on almost all chromosomes, except chromosomes 14 and 18 . We observed the highest number of DMRs on chromosomes 9 and 19  of the DMRs located within gene bodies, mostly intronic regions (n = 40). Furthermore, we observed that DMRs were enriched in transcription factor binding sites . These TFBS were mostly downstream to gene promoters, except six, which were located in the promoters of TGFBR3, RGPD8, HTRA3, LCN10, FAM174B, and IL17C . We next performed TFBS enrichment analysis of the 122 transcription factors (TFs) included in the ENCODE Uniform TFBS track [SF3B1mut cases develop from. However, the highest odds ratio was identified for the histone deacetylase HDAC6 with two DMRs at the promoter regions of HTRA3 and FAM174B. HDACs’ role in the initiation and progression of cancer has been extensively studied, as reviewed in [XXYLT1, HTRA3, and ARID3A genes. Eleven DMRs (16%) were located at ten unique promoter regions  and two genes (HTRA3 and FAM1748) had higher expression and hypomethylated promoters in CLL patients with SF3B1mut compared to SF3B1WT  containing three of the genes  as significantly affected by differential methylations  between LP- and IP-CLL among the SF3B1WT CLL samples  showed stable methylation profiles among the SF3B1WT CLL samples  and hypermethylated ; and non B cell-specific sites hypomethylated in CLL  and hypermethylation in CLL . The CLL-specific CpGs are expected to be associated with the tumorigenic transformation to CLL [SF3B1mut compared to SF3B1WT patients is related to the normal B cell differentiation process, and that the other part is specific to SF3B1mut CLL.In addition, to further investigate if the observed DMRs between ramework . The autn to CLL . Eight  has been shown to impact methylation in acute myeloid leukaemia [SF3B1mut. To acquire additional insight into the methylome differences in CLL patients with and without SF3B1 mutations, we analyzed genome-wide methylation profiles using MeDIP-seq. We identified 67 regions with significantly lower methylation levels in SF3B1mut CLL  have been significantly associated with SF3B1mut CLL patients [EHMT1 and UCKL1 genes were the only genes that overlapped the DMRs reported here, and were listed as genes with altered splicing associated with SF3B1 mutation in CLL patients [EHMT1, in particular, has also been associated with DNA methylation [SF3B1mut CLL patients.The question remains what the cause for the altered methylation pattern might be. So far, there are no reports on the patients , and werpatients . Althougpatients  and DNA patients . Furtherhylation ,63 and, IGHV mutational status and allowed to clearly separate the samples by the SF3B1 mutational status  and intermediate- (IP-CLL) programmed CLLs, and the role of IKAROS in B cell development has been highlighted in the early stages [In agreement with previous studies, most of our DMRs were located in genic regions B. The geelopment  and has elopment . IKAROS elopment . Moreoveelopment ,73, a drelopment . Of noteelopment ,36. The  B cells ,52,53. Iy stages ,76,77,78SF3B1mut patients at IKAROS binding sites and the enrichment of the hypomethylation sites in weak enhancers is noticeable and requires further investigation.Induction of IKAROS in CLL cells is associated with poor disease outcome  and promp.L162R [IKAROS family proteins interact with nucleosome remodeling and deacetylase (NuRD) and PRC2 . In T cep.L162R , emphasiHTRA3 and FAM174B. Although FAM174B has binding sites for more TFs, HTRA3 has binding sites in this region only for HDAC6 and the PRC2 component, SUZ12. Histone deacetylase inhibitors have been already in phase II clinical trials to treat patients with breast cancer [BCL9L gene—an activator of Wnt signaling associated with B cell malignancies that have been implicated in cancer development and epithelial-mesenchymal transition through a down-regulation of c-Myc, cyclin D1, CD44, and vascular growth factor in tumor cells [SF3B1 mutation has been previously reported [BCL9L hypomethylation in poor prognosis of the CLL patients with SF3B1mut should be further investigated.Although significant, but with only two DMRs in TFBSs, is the histone deacetylase, HDAC6. The hypomethylated two sites for this region were located within promoter regions t cancer . Furthert cancer ,87,88. Aor cells . Hypometreported  and it hreported . Interesreported ,87,88. AIn comparison with previous studies, our MeDIP-seq approach covers a broader part of the genome. For example, 450-K methylome arrays analyze 482,486 CpGs, most of which are located in genic regions and CpG islands . Since tSF3B1mut CLL samples were classified as LP- and IP-CLL subtypes [p-value = 0.09), it is in agreement with other studies, which reported the highest occurrence of CLL with SF3B1mut in naïve B cell-like CLL (LP-CLL), and lowest in memory B cell-like CLL (HP-CLL) [SF3B1 mutations are, at least in part, associated with B cell developmental stages  ,52,53. I(HP-CLL) ,36, indil stages . Some ofTP53, SF3B1, ATM, XPO1, and NOTCH1 mutational status were analyzed by a PCR panel followed by next-generation sequencing, as described in [SF3B1, TP53, and ATM was analyzed, and XPO1 exons 12, 13, and 15 were evaluated. IGHV mutational status was determined, as previously described [Clinical information of the patients is summarized in ribed in . In partescribed . PeripheThe integrity of DNA was evaluated on a 1% agarose gel and 1.3 µg was subjected to MeDIP. Methylated DNA immunoprecipitation (MeDIP) was based on a method developed by  with modMeDIP sequencing reads were aligned to the hg38 reference genome (Genome Reference Consortium GRCh38) with BWA v0.7.15 aln followed by samse modules . Patientp-values were corrected for multiple testing by false discovery rate (FDR) [MeDIP-seq reads were processed in R with QSEA v.1.14.0 , accordite (FDR) . We conste (FDR) , and CGIte (FDR) , transcrte (FDR) ,40,41,42te (FDR) ,50 were te (FDR) . PromoteSF3B1 mutational status information. Differentially methylated probes were called using lmFit and eBayes functions of limma [p-value and fold change thresholds applied for the MeDIP-seq data, as described above.Methylation microarray data from 196 CLL patients’ samples (CLLmethylation) covering 435,155 CpGs  were obtained via BloodCancerMultiOmics2017 R packages  with Expp-value was < 0.05. To identify possible genes affected by the differential methylation of weak enhancers, no threshold on log2(fold change) was set.RNA libraries were generated using the TruSeq Stranded Total RNA sample preparation kit  which includes a Ribo-Zero depletion of ribosomal RNAs prior to library preparation. Sequencing of 50 bp paired-end reads was performed on a HiSeq2000 . RNA-seq reads were mapped to the same reference with STAR v2.6.0c  and GENCSF3B1mut.We selected 16 DMRs with |log2 fold change| > 2 for validation by the EpiTYPER. Primers were designed for CpGs within 16 DMRs. We used a subset of six CLL samples with and six CLL samples without For the validation of the differentially methylated regions identified by MeDIP-seq, we used the EpiTYPER assay, which is based on bisulfite conversion followed by PCR amplification using one primer containing a T7 promoter sequence, followed by in vitro transcription and Uracil-specific cleavage of the RNA. Fragments were then analyzed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF) mass array analysis ,104.www.epidesigner.com accessed on 24 March 2021) and purchased from Integrated DNA Technologies . Oligo sequences, genomic coordinates, and annealing temperatures are given in M.SssI (CpG) methyltransferase . Standards of 0%, 20%, 40%, 60%, 80%, and 100% were generated by mixing WGA DNA with WGA and in vitro-methylated DNA. For each assay, a methylation standard was run in parallel. Methylation for individual CpG units was calculated with the EpiTYPER 1.3 software. Subsequently, methylation values (0 to 1) for a given region were calculated as the mean of the analyzed CpG units that passed the quality criteria. CpG units were excluded from the analysis when: (i) less than 50% of all samples had values; (ii) CpG units within one amplicon had an identical mass; and (iii) > 3 CpGs within one CpG unit. DNA methylation values for the amplicons are given in Primers for the EpiTYPER assay were designed using the online tool EpiDesigner with default settings  within their gene bodies or promoter regions and subjected to functional enrichment analysis with gProfiler2 with default options (version e103_eg50_p15_eadf141) ,106.SF3B1 mutation compared to CLL without SF3B1 mutation were used as input for findMotifsGenome.pl module of HOMER v4.11.1 [Differentially methylated 250-bp regions in CLL patients with  v4.11.1  with theFor this part of analysis, we remapped samples to the hg19 reference genome (Genome Reference Consortium—GRCh37) and ran the QSEA R package v.1.14.0 , as befo