Effect Of Silencing P27RF-Rho Expression On The Biological Behavior In Human Non-small Cell Lung Cancer Cell A549

Background: Non-small cell lung cancer(NSCLC)accounts for 80% to 85% of all lung cancer cases.It has high incidence,high mortality,easy transfer and poor prognosis.At present,five years after the diagnosis of NSCLC patients survival rate of less than 15%,although in recent years new treatments,but there is no significant effect of treatment of NSCLC.The invasion of tumor cells and the transfer of other organs is a problem and a key to the treatment of lung cancer.Therefore,it is very important to find a new therapeutic method which can inhibition the metastasis and invasion of lung cancer cells.The latest study found a P27kip1-related protein,P27RF-Rho played an important role in tumor metastasis and invasion.P27RF-Rho mainly through the regulation of Rho pathway to activate Rho protein,thereby promoting tumor evolution and progression.P27RF-Rho as a cancer gene,its in the process of the development of lung cancer and the role of the corresponding molecular mechanism is still a lack of in-depth research.RNA interference(RNAi)is a high specificity method for silencing the target gene,and has been widely used in anti-tumor,anti-virus and cell signal transduction pathway.The lentiviral mediated RNAi is the most commonly used methods.Objective: In non-small cell lung cancer,we silence the P27RF-Rho gene of the cell line A549,observe its influence of the cell proliferation,cell cycle,apoptosis,migration and invasive ability,to explore the feasibility of its as a new potential sites for lung cancer targeted therapy.Method:1.Construction of P27RF-Rho RNAi lentivirus and infection of A549 cells.The A549 cells were divided into P27RF-Rho-si RNA group,Scramble-si RNA negative control group and A549 blank control group.Westem blot was used to detect the expression of P27RF-Rho.2.Cell proliferation experiments: MTT colorimetric method was used to detect cell proliferation.3.The expression of P16 m RNA was significantly higher than that of the negative control group and the blank control group,but the expression of CDK5 m RNA was decreased significantly.The Western blot result showed that the expressions of P27kip1 significantly higher and Cycline protein were significantly decreased compared with the Scramble-si RNA negative control group and the blank control group.4.Cell apoptosis assay: Western blot was used to detect the expression levels of p53,Bax and anti-apoptotic protein Bcl-2.5.Cell migration experiments: cell scratch test was used to detect cell migration.6.Cell invasion experiment: Transwell assay was adopted to evaluate the invasive ability of A549 cell.Result: 1.The lentiviral vector containing P27RF-Rho interference sequence was successfully constructed and the stable cell lines of P27RF-Rho-si RNA were established.The Westem blot result showed that the protein expression of P27RF-Rho was significantly decreased.2.The cell proliferation ability of the experimental group was significantly lower than that of the negative control group and the blank control group.3.The expression of P16 m RNA was significantly higher than that of the negative control group and the blank control group,but the expression of CDK5 m RNA was decreased significantly(P <0.05).The Western blot result showed that the expressions of P27 and P27kip1 protein were significantly decreased compared withthe Scramble-si RNA negative control group and the blank control group(P <0.05).4.The expression levels of P53 and Bax protein in the experimental group were significantly higher than those in the negative control group and blank control group,while the anti-apoptotic protein Bcl-2 decreased.5.The cell migration ability of the experimental group was significantly lower than that of negative control group and blank control group.6.The cell invasion ability of the experimental group was significantly lower than that of the blank control group and the negative control group.Conclusion: 1.Silencing P27RF-Rho gene can inhibit the proliferation of non-small cell lung cancer cell line A549.2.Silencing P27RF-Rho gene can promote the cell apoptosis of non-small cell lung cancer cell line A549.3.Silencing P27RF-Rho gene can inhibit the migration and invasion of non-small cell lung cancer cell line A549.