PRMT5 Regulates Proliferation And Apoptosis Through NF-?B Signaling In Bladder Cancer

Objective:Bladder cancer is one of the most common urinary cancers,with the increase of new cases and deaths each year.The diagnosis of bladder cancer is mainly based on bladder examination and urinary cytology examination.The sensitivity of diagnosis is low,so many patients in the early stages of bladder cancer is difficult to detect.Exploring the relationship between gene expression changes and the development of bladder cancer from the gene level may provide new directions for bladder cancer diagnosis and the development of targeted drugs.And it could also providea new thought for finding non-invasive,sensitive and specific diagnosis methods.Protein arginine methyltransferase?PRMT?is involved in a number of physiological processes.PRMT5 is a typical type II PRMT enzyme and has a conservative methyl transfer enzyme catalytic structure.PRMT5 is widely distributed in many types of cells,and its content levels vary from cell to cell.The methylation modification saturized by PRMT5 plays an important role in many cells,and its abnormal expression affects the development of multiple types of malignant tumors.PRMT5 plays its biological function in two main ways.The one is the expression of epigenetic regulatory target genes and the other one is to directly modify the key signaling molecules in cells through methylation.In this study,we explored the expression pattern and clinical pathological significance of PRMT5 in bladder cancers.The effects of PRMT5 expression on biological behaviors in bladder cancer cells,and the potential molecular mechanisms were analyzed by experiments in vivo and vitro.Methods:1.Immunohistochemistry:Eighty cases of bladder cancer tissue?the pathological type is transitional cell carcinoma?and 10 cases of adjacent normal tissue were taken from patients who underwent bladder cancer surgery from the Fourth Affiliated Hospital of Chinese Medical University in 2016-2017.The samples were buried in paraffin and slice was prepared.Slice was incubated with PRMT5 at 4?overnight.Slices were incubated with biotinase-labeled secondary antibody at 37?for1 hour.After incubation,the slices were developed through DAB method.2.Quantitative PCR:Total RNA was extracted using the RNeasy Mini kit.cDNA was prepared by the SuperScript III First-Strand Synthesis Kit.qPCR was performed using SYBR Green MasterMix.3.Cell culture:The cell lines used in this experiment are listed as follows:human normal bladder epithelial cells SV-HUC-1,human bladder smooth muscle cells HBSMC,human bladder cancer cell T24,UM-UC-3,BIU-87,J82,RT4,and 5637.DMEM and PRIM-1640 containing 10%fetal bovine serum were used for cellculture.Cells were incubated at 37?,5%CO₂ and saturated humidity.4.Western blot:Total protein was isolated by SDS-PAGE and transferred to the PVDF membrane.Nonspecific blocking is performed using BSA.Antibodies used were:PRMT5,Cleaved-Caspase3 and GAPDH.5.Cell transfection:PRMT5 shRNA is purchased from DHARMACON and transfected using Lipofectamine and plus reagent.Puromycin was used for cell screening.PRMT5 siRNA is purchased from DHARMACON and transfected using Lipofectamine RNAi MAX reagent.PRMT5 plasmid is purchased from Origene and transfected using Lipofectamine 3000.6.Cell proliferation assay:1×10⁶cells were seeded in 10cm dishes in RPMI 1640containing 10%FBS.Cell numbers were counted in 2 and 4 days.For colony formation assay,50/well cells were placed in six-well plates in RPMI 1640 containing10%FBS for 14 days.Colonies were fixed with methanol and stained with 0.1%crystal violet in 20%methanol for 15 min.7.Annexin V apoptosis assay:The transfected cells were collected and rinse once with PBS buffer after centrifugation.After resuspending the cells,add Annexin V-FITC and PI staining reagents.The cells were incubated at room temperature in dark for 15 minutes.Apoptosis levels were detected using the flow cytometer.8.JC-1:The transfected cells were collected and resuspended with no serum media after low-speed centrifugal.The control group was pre-dyed for 5 minutes using CCCP,and cells were incubated with JC-1 dye at 37?,5%CO₂ for 45 minutes.The cells were collected and resuspended with PBS buffer.Mitochondrial membrane pitential level was detected using the flow cytometer.9.Luciferase assay:p5XIP10 was transfected in T24 cells using Lipofectamine and Plus Reagents.48 hours later,the NF-?B luciferase activity was monitored.10.Chromatin immunoprecipitation?ChIP?:The truChIP Chromatin Shearing Kit was used to prepare chromatin.Chromatin was sheared into 200-to 700-bp fragments using a Covaris S2 instrument.The IgG and NF-kB p65 antibodies were used for immunoprecipitation by the Quick Chip Kit.SYBR Green Master Mix was used for qPCR to quantify precipitated DNA.11.Xenograft study:1×10⁶ T24 cells were injected subcutaneously into C57BL/6J mice in the right flank.Tumor volume was measured.EPZ015666 or control vehicle?PBS?was administered by gavage twice daily?50 mg/kg?from 14days after injection.Mice were sacrificed 28 days after injection.12.Statistical analysis:Data were analyzed using GraphPad Prism 7.The statistical difference was calculatedby using two-tailed Student t test or one-way ANOVA.A P-value below 0.05 was considered significant.Results:1.PRMT5 was up-regulated in bladder cancer tissue and cells.PRMT5 was highly expressed in bladder cancer tissue.36 of the 80 cases of bladder cancer tissue showed high expression of PRMT5,and only 2 of the 10 cases of normal cancer side tissue were PRMT5 positive stained.Of the 10 pairs of samples,PRMT5 expression was higher in 8 of the tumor tissue than that of adjacent normal tissue at protein levels.At mRNA levels,PRMT5 expression was higher in 10 cases of tumor tissue than that in normal tissue.PRMT5 was higher in bladder cancer cell lines than that in normal bladder cells.Analysis of PRMT5 RNA seq data and clinical pathology information in TCGA database showed that patients with high PRMT5 expression had shorter survival times.And PRMT5 expression was associated with tumor pathology T grade in patient tissue.2.PRMT5 promoted proliferation of bladder cancer.The number of cells and colonies was significantly reduced after PRMT5 depletion in T24 and UM-UC-3 cells,and restored partly after PRMT5 plasmids transfection.In contrast,the number of cells and colonies was increased when PRMT5 overexpression in BIU-87 and 5637cells,while was decreased after siPRMT5 transfection.3.PRMT5 suppressed apoptosis of bladder cancer.Level of apoptosis increased while mitochondrial membrane potential decreased in T24 cells transfected with si PRMT5.In contrast,apoptosis level decreased and MMP increased in BIU-87 cells trasnsfected with PRMT5 plasmid.PRMT5 overexpression downregulated the expression of Cleaved-caspase 3.4.PRMT5 activated NF-?B signaling pathway in bladder cancer.The activity of NF-?B was reduced in T24 cells transfected with shPRMT5.NF-?B activity was reduced in T24 cells treated with EPZ015666 in a does-dependent manner.The levels of BCLXL and c-IAP1 were decreased after the inhibition of the expression or activity of PRMT5 in bladder cancer cells.Chromatin immunoprecipitation?ChIP?results showed that pharmacological inhibition of PRMT5 by EPZ015666significantly impairs NF-?B p65 occupancy on c-IAP1 and BCLXL pro-moters.5.PRMT5 promoted bladder cancer proliferation in xenograft.Bladder tumor model in mice was build and take the PRMT5 inhibitor EPZ015666 by gavage.The tumor volume and weight of the gavage EPZ015666 group of mice were significantly smaller than the control group.In addition,the expression of BCLXL and c-IAP1 in bladder tumor tissue decreased significantly.Conclusion:PRMT5 is up-regulated in human bladder cancer tissues and cell lines.PRMT5 overexpression promotes the proliferation,while PRMT5 knockout maintains the mitochondrial membrane potential and induces apoptosis in bladder cancer cells.PRMT5 activates the NF-?B pathway in bladder cancer cells and increases the expression of its target genes BCLXL and c-IAP1.In the mouse bladder tumor model,PRMT5 inhibitor?EPZ015666?inhibits tumor growth and the expression of BCLXL and c-IAP1.