Study Of The Effect On Biological Behavior Of Bladder Cancer Cells By MiR-520b And Its Molecular Mechanisms

[Background]Bladder cancer is one of the most common human malignancies, and its pathogenesis is complex, involving a large number of genes’ expression, aberrant functions and changes in multiple signaling pathways. At present, the molecular genetics of development and progression of bladder cancer is still unclear.MicroRNA is a class of small noncoding single-stranded RNAs found in eukaryotes, about21to25nucleotides in length, which usual regulates gene expression at the post-transcriptional level. A large number of studies have shown that microRNA participate in tumor cell proliferation, differentiation, invasion and apoptosis. Accumulating evidences indicate that aberrant expression of microRNA can contribute to bladder tumorigenesis. miR-520b is a newly discovered abnormal expression microRNA in tumors, which has been found in a low level expression in a variety of malignancies, so we hypothesized that it function as a tumor-suppressive gene. [Aims]Our research focused on discussing the biological function and molecular mechanisms of miR-520b in the pathogenesis of bladder cancer.[Methods]1) miR-520b levels were assessed by real time RT-PCR, the objects consisting of human bladder cancer cell lines (T24, UC3), immortal human bladder mucous epithelial cell SV-HUC-1, eighteen primary bladder cancer tissues and their pair-matched adjacent non-tumorous bladder mucosal tissues from patients.2) To better characterize the role of miR-520b in bladder cancer, upregulation of miR-520b expression were conducted by transfecting bladder cancer cell line T24and UC3with chemically synthesized miR-520b mimic. In vitro experiments, including CCK8assay, plat clone formation assay, and flow cytometry analysis (FCM) were performed to validate that miR-520b had an effect on proliferation of bladder cancer cells. Wound healing assays and transwell chambers were used to evaluate the effect on cell migration and invasion.3) Aided by informatics analysis, we predicted that the probable genes targeted by miR-520b and classified them based on functions. Then according to previous studies and pre-experimental results, we concluded that probably miR-520b targeted Cyclin D1. The target gene was then determined by luciferase assays, quantitative RT-PCR and western blot.[Results]1. There were16(88.9%) cancer tissues presented with miR-520b in down-regulated pattern while comparing with normal adjacent tissue. The mean miR-520b expression level in cancer tissues was about56%of that in normal adjacent tissue. However, no difference was detected when the bladder cancer cases were stratified by histological stage or grade.2. miR-520b was found down-regulated in bladder cancer cell T24and UC3. The mean miRNA expression level in two cancer cell lines was about1/2of that in SV-HUC-1.3. We found that over expression of miR-520b could induce G1-phase arrest in T24and UC3cells, and subsequently inhibit T24and UC3cells growth and colony formation.50nM miR-520b mimic demonstrated a potent inhibitory effect at48h after transfection. It could reduce cell viability by31.8%in T24and34.0%in UC3. Also, the colony formation ability was impaired after miR-520b mimic treatment, with a7.5%drop in T24cell and10.6%in UC3cell in vitro colony formation rates. However, no effect on cell apoptosis was detected. The cell migration and invasion were undisturbed either.4. We identified that Cyclin D1, CDK4, pRb and E2F3could be down-regulated by miR-520b. Moreover, as a novel target, CCND1was verified as one of the key mediator of miR-520b induced G1-phase arrest in T24and UC3cells. Further qRT-PCR analysis and luciferase assay confirmed that miR-520b transcriptionally repressed Cyclin D1by interacting with the essential binding sequence located in3’-UTR of Cyclin D1.[Conclusion]miR-520b was frequently down-regulated in bladder cancer both in two bladder cancer cell lines, T24, UM-UC-3and in clinical samples. miR-520b suppresses bladder cancer cell proliferation, inhibits colony formation and induces Gl-phase arrest.CCND1was a novel direct target of miR-520b, and it played an important role in the anti-tumor mechanism of miR-520b.