Effects And Mechanism Of Small Interfering RNA Targeting Fer On The Biological Behavior Of Bladder Cancer T24 Cells

Objective and background:Bladder cancer is the most commonly tumor in urinary system in China,In 2012,an estimated 73,510 new cases will be diagnosed,which could result in an estimated 14,880 deaths.Bladder cancer is characterized by easy recurrence and strong invasiveness,and the exact mechanisms of tumor formation and progression are not yet completely understood,so the available protocols are far from satisfaction.Therefore,we must understand biological behavior of bladder cancer, and Search for biological markers feasible and to investigate the therapeutic target.Feline sarcoma-related protein(Fer)is a nuclear and cytoplasmic non-receptor protein tyrosine kinase that is extensively expressed in human cells.Fer has been reported to be upregulated in many Malignant tumors,which is connected with various biological processes such as cell proliferation, migration,and invasion,and regulate cell signal transduction by various growth factor receptors.However,no literatures have reported the clinicopathological characteristics and molecular mechanisms of Fer expression in bladder cancer.Mitogen activated protein kinases /Activator protein-1(MAPK/AP-1) signaling pathway is the classic celluar signal transduction pathways, which regulates cell proliferation,differentiation,survival,transformation and apoptosis. In this study,we initially investigated the overexpression of Fer in bladder cancer,then we use si RNA silenced the production of Fer gene in human bladder cancer T24 cells,and found the effection of Fer on biological behavior and MAPK/AP-1 signaling pathway of T24 cells.It provides a new way for clinical treatment of bladder cancer,and it lays theoretical foundation.Methods: We investigated the expression of Fer in bladder cancer tissues and cells by q RT-PCR, western blot analysis and immunohistochemistry. Chemical synthesis in Fer small interference RNA, through the liposome transfection method transfect the si RNA into T24 bladder cancer cells. Detect knockout efficiency of Fer by q RT-PCR and Western blot technical. We found that cell proliferation and cell cycling was inhibited by MTT assay and Flow cytometry, cell migration and invasion were suppressed by Cell wound healing assay and Transwell assay. Lastly, we definited the involvement of MAPK/AP-1 pathways by Western blot analysis.Results: We indicated that Fer expression was overexpressed in bladder cancer tissues and cells compare to adjacent normal bladder tissues and cells. Clinicopathological analysis showed that Fer expression was significantly concerned with tumor stage, histological grade, and lymph node status(P <0.05). Fer si RNA could significantly inhibit the Fer expression of T24 cells. MTT techniques revealed the cell Proliferation inhibition rate in RNAi(Si)group was 22.9%、24.4% and 42.5% at 24, 48 and 72 h respectively,which was significantly inhibited as compared with the blank control(Con) group and negative control(Nc) group(P<0.05).Flow cytometry revealed the percentage of G0/G1 and S phase cells in RNAi(Si) group was(52.34±2.049)% and(35.04±3.810)% respectively, which were significantly different from those in the blank control(Con) group and negative control(Nc) group(P<0.05). Fer si RNA also suppressed proliferation, migration and invasion of T24 cells as compared with the blank control(Con) group and negative control(Nc) group. Meanwhile, the phosphorylation levels of MAPK / AP-1 signaling pathway were significantly reduced. as compared with the blank control(Con) group and negative control(Nc) group.Conclusion: The overexpression levels of Fer in bladder cancer tissues and cells are related to the tumor stage, histological grade of bladder cancer, and plays a significant role in the development of bladder cancer. Fer probably can be used as a useful molecular marker for prognosis of transitional carcinoma of bladder. The knockdown of Fer expression by si RNA caused G0/G1 phase arrest, and suppressed proliferation, migration and invasion of T24 cells, might provide a genetic locus in bladder cancer targeted therapy. The inactivation of MAPK/AP-1 signal pathways might be one of the molecular mechnisms that Fer si RNA affects biological behavior of T24 cells.