The Anti-proliferation Role And Mechanism Of MiR-877-3p Induced P16 Activation In Bladder Cancer

Urinary bladder cancer(BCa)is the most common urogenital malignant tumor.It threats the life of the patients and causes burdens of our society,which has become a serious issue of public health.Bladder cancer is a kind of epithelial tumor and characterized by locally invasion and metastasis.Despite the development of the treatment,which including surgery,chemotherapy,radiotherapy and the combination of various treatments,the prognosis and long-term survival rate remain disappointing.The occurrence and progression of bladder cancer are related to the overactivation of oncogene,as well as the silence of tumor suppressor gene.Therefore,to restore the expression and function of tumor suppressor gene has its unique value in the research of target therapy in bladder cancer.MicroRNAs(miRNAs)are small endogenous,non-coding RNAs with approximately 19～22 nucleotides.The achievement of their function is through the up-regulation or down-regulation of the coding genes by targeting the specific sequence.Numerous researches have shown that the aberrant expressions of miRNAs are involved in the occurrence and progression of bladder cancer.Our research aimed to demonstrate the activation of tumor suppressor gene p16 by miR-877-3p in bladder cancer and its probable molecular mechanism,as well as to explore the function of miR-877-3p in bladder cancer.The main investigations and results are as follows:1)p16 is down-regulated in bladder cancer cells.We measured the expression of p16 in three bladder cancer cells lines(T24,UM-UC-3 and 5637)with real-time RT-PCR.It turned out that all three bladder cancer cell lines exhibited a lower expression level of p16 compared with SV-HUC-1 cell line(a transitional epithelial cell line).2)We scanned 1.5kb of p16 promoter to search for probable complementary site of known human miRNAs by online prediction tool and chose miR-877-3p as our research object.Real-time RT-PCR was performed to quantify and analyze the expression levels of miR-877-3p in three kinds of bladder cancer cell lines(T24,UM-UC-3 and 5637 cell lines)versus SV-HUC-1 cell.We observed that compared with SV-HUC-1 cell line,miR-877-3p in three bladder cancer cells showed significant lower expression pattern.Moreover,it is worth noting that treatment with 5-Aza-2’-deoxycytidine(5-Aza-CdR)could increase the expression of miR-877-3p in bladder cancer cells.3)miR-877-3p which targeted the-320～-299 site of the p16 promoter could up-regulate the p16 expression in bladder cancer cells on both mRNA and protein levels.The sequence integrality of miR-877-3p is essential in the miR-877-3p induced p16 activation.Luciferase assays showed higher p16 promoter activity with the treatment of miR-877-3p.And Chip assay confirmed that the activation of p16 was due to the direct interaction between miR-877-3p and p16 promoter.4)We performed gain-of-function study to explore the role of miR-877-3p in bladder cancer cells.The results showed that overexpression of miR-877-3p could inhibit the proliferation in vitro as well as the tumorigenicity in vivo.Meanwhile,analysis by FACS pointed out that miR-877-3p could induce Gl-phase arrest.5)RNA interference(si-pl6)was used to knockdown the expression of p16 in bladder cancer cells.The application of si-p16 abolished the miR-877-3p-mediated cell proliferation inhibition and cell cycle arrest significantly,which indicated that the activation p16 was mainly responsible for the anti-proliferation role of miR-877-3p in bladder cancer cells.In summary,our study presented the anti-proliferation function of miR-877-3p in bladder cancer cells through activating the expression of p16 by directly targeting the specific promoter site.