| Background and Objective:Human bladder cancer was the ninth most common cancer with an estimated 429,793 new cases and 165,068 deaths registered in 2012 worldwide. It was three times more common in men compared with women and ranks sixth among cancers in men. Bladder cancer comprises distinct histopathological patterns and clinical behaviors and thus making serious obstruction in the controlling of bladder cancer. Long noncoding RNA 19 (H19) has been shown to promote bladder cancer cell proliferation and metastasis. However, little is known about how miR-675, mature product of H19, contributes to bladder cancer cell proliferation. The aim of this article is to study the expression of miR-675 in bladder cancer and investigate its role in bladder cancer cells and its possible mechanisms, and provide new target for the treatment of clinical bladder cancer.Methods:The expression of miR-675 in bladder cancer tissues and adjacent normal tissues, healthy urinary bladder tissues and bladder cancer cell lines was studied by using qRT-PCR. Next, we constucted pcDNA-H19 plasmid that overexpress H19 and designed H19-siRNA that knock-down the expression of H19, and transfected them into 253 cells, the effect of the expression of H19 on the bladder cancer cell proliferation and expression of miR-675 was investigated by cell proliferation assay and qRT-PCR. As for miR-675, we conducted further research, miR-675 minic, miR-675 inhibitor and a negative control was transfected into 253J cells and RT4 cells, and the effect of miR-675 on cell biological behavior including cell proliferation, call apoptosis and cell cycle was detected by cell proliferation assays, apoptosis assays and cell cycle assays. Finally, miR-675 minic, miR-675 inhibitor and a negative control was transfected into 253J cells, and western bloting was used to explore the role of miR-675 on apoptosis-related protein p53, Bcl-2 and Bax and cycle-related proteins cyclin D1, which affected the mechanism of bladder cancer cell proliferation.Results:(1) The expression of miR-675 was significantly up-regulated in most bladder cancer tissues, however, this increase was not observed in patients without bladder cancer. Furthermore, increased expression of miR-675 was also observed in several human bladder cancer cell lines (RT4, HT-1376,5637,253J, TCCSUP, T24 and J82). Thus, these data indicate that up-regulation of miR-675 may be related to the progression of bladder cancer.(2) Ectopic expression of H19 caused a significantly up-regulation of miR-675 expression and increased cell proliferation of 253J cells and RT4 cells. Furthermore, H19-siRNA treatment decreased the miR-675 expression level, and inhibited 253J cell proliferation.(3) miR-675 expression was significantly enhanced or decreased with the transfection of miR-675 mimic or inhibitor into 253J cells, and more importantly, overexpression of miR-675 enhanced cell proliferation of 253J cells and RT4 cells, while miR-675 inhibitor showed a significantly inhibitory role in cell proliferation.(4) Cells in the S phase were significantly increased, and cells in the G0/G1 phase were significantly decreased by the miR-675 mimic treatment in 253J cells and RT4 cells. And miR-675 inhibitor significantly promoted the apoptosis of bladder cancer cells.(5) miR-675 significantly decreased p53 expression, as well as p21, furthermore, miR-675 enhanced cyclin D1 expression. miR-675 mimic markedly down-regulated the expression ratio of Bax/Bcl-2, and miR-675 inhibitor significantly enhanced the expression ratio of Bax/Bcl-2.Conclusion:miR-675 expression is markedly increased in bladder cancer tissues and cell lines compared with normal control. miR-675 was positively regulated by H19 and overexpression of miR-675 increases cell proliferation of human bladder cancer cells. We further confirmed that miR-675 induces a G1 arrest and inhibits cell apoptosis, and this may depend on its negative regulatory role for p53 expression. Taking together, our results suggest an important role for miR-675 in the molecular etiology of bladder cancer and highlight potential application of miR-675 in bladder cancer therapy. |
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