The Role Of MiR-221on Immune Escape Of Bladder Cancer Cells By Silencing PUMA

Bladder cancer is the most common malignancy of the urinary tract. Accordingto the latest statistics of the U.S. National Institutes of Health (NIH),73,510newcases of bladder cancer were diagnosed in2012,14,880of which died from thedisease. The incidence of bladder cancer is increasing in our country. In thedevelopment and progression of bladder cancer, the cancer cells and the host undergothree stages: immune surveillance, immune balance and immune escape. Cancerimmune escape makes the clinical treatment of patients with bladder cancer morecomplex. Therefore, the study of bladder cancer immune escape mechanism has beenan important research direction in the field of urologic oncology in recent years.The process of immune cells killing tumor cells involves the immune cellspassing apoptotic signals to tumor cells, as well as the apoptotic signal transductionwithin tumor cells. However, the vast majority of the tumor cells escape immunestudy are focused on passing the apoptotic signal hindered immune cells to the tumorcells, such as deletion of the tumor cell antigen, blockage of tumor cellsantigen-presenting process, tumor cells secreting immunosuppressive factor, andchange of tumor cell epigenetics. Nevertheless, studies have shown that immune cellsto tumor cells pass apoptotic signaling pathway is not completely blocked.Theimmune escape of tumor cells can still partially acceptapoptosis signal coming fromimmune cells. Therefore, we assume that the apoptotic signal transduction in bladdercancer cell disruption may be an important factorwhichinduces bladder cancer cellimmune escape. Intracellular signal regulation is extremely complex, and nearly10years of research found that small RNA (microRNA or miRNA) plays an importantrole in the regulation of intracellular signal. The control mechanism can besummarized as the following: the mature miRNA silencing complex assembly andidentification of target mRNA through complementary base pairing, the degradationof target mRNA or inhibition of target mRNA translation according to the degree ofcomplementarity of the different guidance silencing complex to regulate intracellularsignal transduction. The study was conducted based on previous experimental data which have shown that the expression of miR-221of bladder cancer was significantlyhigher than normal bladder tissue, to study the role of miR-221targeting PUMA genein bladder cancer immune escape.Objective: To investigate miR-221silencing, the effect of PUMA in immune escapesin bladder cancerMethods: We detected the cell growth after transfected with miR-221inhibitor. Weused artificial synthesis miR-221inhibitors (has-miR-221inhibitor), NegativeControl FAM. The cells were divided into three groups:①Transfection of miR-221inhibitor②Negative Control③control group, transfected withLipofectamineTM2000miR-221inhibitor, Negative control FAM were transfectedT24,5637, J82cells using MTT assay transfection24h,48h,72h after cellproliferation activity.48h after transfection, using qRT-PCR detection of theexpression of miR-221; RT-PCR and western-blot detection the mRNA and proteinexpression levels of miR-221target genes PUMA and apoptotic gene Bax, Bcl-2;flowcytometry detected the change of apoptotic rate, AO-EB staining detected apoptosismorphological change.Invasive detection of cell changes in bladder cancer cells transfected withmiR-221after48hours. Human bladder transitional cell carcinoma T24,5637, J82cells were divided into①transfected with miR-221inhibitor group,②negativecontrol group,③control group, and were transfected48h, using RT-PCR andWestern-blot detection the mRNA and protein expression changes of MMP-9,MMP-2, VEGF-C. And Aggressive change of cells was detected with transwell.Results:1.Transfection of miR-221inhibitor to human urothelial carcinoma cells5637J82T24，promote apoptosis.①The transfection efficiency is about80%under fluorescence microscopy aftertransfection of miR-221inhibitor for48h.②Transfection24h,48h,72h after, MTT results showed: proliferative activityof three kind of cells in the experimental group were significantly lower, theinhibition rate increased, and in a time-dependent manner, the proliferative activity ofnegative control group and the control group inhibition rate have not changed significantly.③Transfection48h after, qRT-PCR detection expression of miR-221in threekind of cells: the expression level of experimental group of miR-221compared to theNC group and the control group was significantly reduced, and the difference wasstatistically significant (P0.05), the NC group and the control group showed nosignificant difference.④RT-PCR,western-blot results showed that: PUMA, Bax mRNA and proteinsexpression levels of the three cells in the experimental group compare with the NCgroup and the control group were significantly increased, and Bcl-2mRNA andproteins expression with the NC group, the control group down, the differences werestatistically significant (P0.05), NC and control groups showed no significantdifference.⑤flow cytometry results are shown: early and late apoptosis rate of the3cells inthe experimental group compared with NC group and the control group weresignificantly increased, the differences were statistically significant (P0.05)， NCgroup and control group was not significant difference, early and late apoptosis rateof5637cells lower than T24and J82cells, which is consistent with the Western-blotresults.⑥AO-EB staining results showed that: the three cells in the experimental groupoccurred apoptosis morphological changes compared with the NC group and thecontrol group in the number of cells was significantly increased, NC group andcontrol group were not significantly different, and5637cells apoptosis morphologicalchanges compared with T24and J82cells in the number of cells occured less, whichis basically the same with flow cytometry and western-blot results, while the NCgroup and the control group showed no significant difference.2. Transfection of miR-221inhibitors reduce human bladder transitional cellcarcinoma invasive①Transfection48hours after, the RT-PCRwestern-blot results are shown:MMP-9/MMP-2/VEGF-C mRNA and proteins expression levels of the three cells inthe experimental group compared with the NC group and the control group wassignificantly reduced, the differences were statistically significant (P0.05), NC and control groups showed no significant difference.②The Transwell result showed that: the number of the matrix film of the threecells in the experimental comparedwith NC group and control group was significantlyincreased, the differences were statistically significant (P0.05), while the NC groupand the control group showed no significant difference.Conclusion:1. MiR-221inhibitor can effectively inhibit expression of miR-221in humanbladder transitional cell carcinoma T24,5637, J82.2. MiR-221inhibitor can inhibit cell proliferation in human bladder transitionalcell carcinoma T24,5637, the J82.3. MiR-221’s down can promote PUAM gene expression increases in T24,5637,J82cells, and further up the pro-apoptotic gene Bax and down the anti-apoptotic geneBcl-2expression, thereby promoting apoptosis.4. MiR-221’s down may promote MMP-9, MMP-2, VEGF-C expression loweredin T24,5637, J82cells, so that the bladder cancer T24,5637, J82cells invasion weredecline.5.The high expression of miR-221can induce bladder cancer cells apoptosisresistance, promote immune escape of bladder cancercells.