Up-regulation Of P21WAF1/CIP1by MiRNAs And The Inhibiting Effects On Bladder Cancer Cells

Part ⅠUp-regulation of p21WAF1/CIP1by miRNAs and the activating mechanismOBJECTIVES:To select the miRNAs which possess higher complementarities with the promoter sequences of p21WAF1/clp1(p21), and investigate the correlations between the selected miRNAs and clinical bladder cancer tissues. Then we test whether the selected miRNAs have the capacities to activate p21expression and preliminarily exploit the activating mechanism.MATERIALS AND METHODS:Target miRNAs complementary to p21gene promoter were predicted by the miRanda algorithm. The miRNAs sequences were retrieved from the miRBase database. The Real-time PCR was performed to assess the expressions of the selected miRNAs and p21mRNA which were extracted from clinical bladder cancer tissues and adjacent non-tumor bladder mucosal tissues. The synthesized dsControl (used as negative control), dsP21-322(used as positive control), miR-103b mimic, miR-370-5p mimic (miR-370mimic), miR-1180-5p mimic (miR-1180mimic) and miR-1236-3p mimic (miR-1236mimic) were transfected into T24and EJ cells with a final concentration at50nM by liposome.72hours after transfection, the p21mRNA was determined by Real-time PCR and the p21protein was determined by Western blot. Furthermore, the subcellular location of p21protein was evaluated by cell immunofluorescence and Western blot. The5’-or3’-biotin covalently linked miRNAs mimics or antisense of dsControl were also transfected into the two cell lines for72hours. The chromatin immunoprecipitation (ChIP) assay was performed to identify whether the biotinylated miRNAs directly interacted with the p21promoter.RESULTS:By the miRanda algorithm,4regions of p21promoter (-907/-887, -552/-537,-397/-379and-243/-226relative to transcriptional start site) were identified with higher complementarities with miR-103b, miR-370, miR-1180and miR-1236. Compared to corresponding normal tissues, miR-103b expression level in tumorous tissues was higher (but without statistical significance), miR-370, miR-1180and miR-1236exhibited significant reduction (P<0.05). In addition, the expressing levels of miR-370, miR-1180and miR-1236positively correlate with p21mRNA levels in tissue samples (P<0.05). And miR-103b appears to negatively correlate with p21expression, but without statistical significance. Transfected all the small RNAs into T24and EJ cells for72hours, PCR analysis showed that dsP21-322, miR-370, miR-1180and miR-1236significantly induced p21mRNA expression. The induction was further confirmed by immunoblot analysis. However, miR-103b failed to up-regulate p21expression at neither mRNA nor protein levels. Moreover, Immunofluorescence showed dsP21-322, miR-370, miR-1180and miR-1236mainly induced nuclear p21protein expression in T24and EJ cells. The subcellular protein was detected by immunobolt, dsP21-322and3miRNAs significant induced p21expression mainly in the nucleus. ChIP assay indicated that miR-370, miR-1180and miR-1236activated p21expression by directly interacting with its promoter.CONCLUSIONS:miR-370, miR-1180and miR-1236were low expressed in human bladder cancer tissues, and positively correlate with p21mRNA expression. The3miRNAs can up-regulate nuclear p21expression by directly interacting with different regions of p21gene promoter. Part ⅡmiRNAs inhibit bladder cancer cells by activating p21expression OBJECTIVES:To investigate the effects of miR-370, miR-1180and miR-1236on bladder cancer cells proliferation, apoptosis, senescence, colony formation, migration and invasion.MATERIALS AND METHODS:Synthesized dsControl, dsP21-322, miR-370mimic, miR-1180mimic and miR-1236mimic were transfected into T24and EJ cells with a final concentration at50nM by liposome. The expression of Cyclin D1, Cyclin-dependent kinase4(CDK4) and CDK6mRNA by standard PCR following transfection at72hours. The PCR products were analyzed on2%agarose gel. Flow cytometry was performed to evaluate the cell cycle distribution and apoptosis of T24and EJ cells. Cells senescence was assessed by β-galactosidase staining.0.5%crystal violet staining was adopted to determine the colony formation of10days’culture for transfected T24and EJ cells. Cells proliferation was evaluated by CellTiter96. AQueous One Solution Cell Proliferation Assay (MTS) at5different time points following transfection (24,48,72,96and120hours). Transwell assay was performed to analyze the migration and invasion abilities of bladder cancer cells. Standard PCR and agarose gel electrophoresis were used to ascertain the p21mRNA expression in both cell lines after co-transfection of miR-1236and siP21(a specific small interfering RNA used to silence p21expression). Moreover, the influences on cell cycle distribution, colony formation and cell proliferation were also analyzed for co-transfection of miR-1236and siP21.RESULTS:Transfection of dsP21-322, miR-370mimic, miR-1180mimic, miR-1236mimic into T24and EJ cells for72hours significantly inhibited Cyclin D1, CDK4and CDK.6mRNA expression, induced cell cycle C0/C1arrest. Further statistical analysis found that compared to Mock and dsControl groups, the cells in G0/G1phase of dsP21-322, miR-370mimic, miR-1180mimic and miR-1236mimic transfected groups were more (P<0.05). In addition, transfection of dsP21-322, miR-370mimic, miR-1180mimic and miR-1236mimic into T24and EJ cells led to more cells in early and late apoptosis than two control groups (P<0.05) and led to more cells senescent. Colony formation assay revealed that compared to two controls, dsP21-322, miR-370mimic, miR-1180mimic and miR-1236mimic transfected cells formed colonies significantly fewer in number and smaller in size. And the corresponding colony formation rates were remarkably lower (P<0.05). MTS assay showed that compared to Mock and dsControl groups, dsP21-322and3miRNAs stably decreased cell proliferation at4days (96hours) later (P<0.05). Migration chamber assay and Matrigel invasion chamber assay indicated that artificial expression of dsP21-322and3miRNAs potently inhibited cells migration and invasion at72hours post transfection (P<0.05). The miR-1236was included in the following experiments based on its lowest expression among the3miRNAs in bladder cancer tissues. The outcomes showed that p21mRNA expression was significantly suppressed after co-transfection of miR-1236and siP21. Besides, knockdown of p21expression significantly abrogated G0/G1cycle arrest, restored the capacity of colony formation and cell proliferation mediated by miR-1236.CONCLUSIONS:Transfection of dsP21-322, miR-370mimic, miR-1180mimic, miR-1236mimic into bladder cancer cells can inhibited Cyclin D1, CDK.4and CDK6expression, and induced cell cycle G0/G1arrest, inhibited colony formation and proliferation. Moreover, miR-1236exerted the inhibiting effects on bladder cancer cells mainly by activating p21expression.