Effect Of Silencing FGF1 Gene Expression By SiRNA On The Invasiveness Of Lung Cancer A549 Cells And Its Mechanism

Objectives: As the current high incidence of malignant tumors,lung cancer is the leading cause of global cancer death-related cases.Non-small cell lung cancer(NSCLC)accounts for 75%-80% of the total number of lung cancer patients.Because the symptoms of lung cancer patients at early stage are cryptic and atypical,most patients are at the advanced stage when diagnosed.Compared with previous radiotherapy and chemotherapy,targeted therapy specifically kills tumor cells by acting on specific sites,the patient’s survival rate is effectively improved,and the side effects are low.Fibroblast growth factor 1(FGF1)is one of the FGF family members.The related studies have found that the high expression of FGF1 is related to the primary lesion size and vascular invasiveness of NSCLC,and the overall survival rate of patients with high expression of FGF1 is decreased,but the specific molecule mechanism is not yet clear.This study was to investigate the effect of silencing FGF1 gene expression by si RNA on the invasiveness of lung cancer A549 cells and its possible molecular mechanism at cellular level.Methods: 1.Design and synthesize FGF1-specific si RNAs(si RNA-1,si RNA-2,si RNA-3,and NC-si RNA)with fluorescent tags.Cells are divided into 5 groups: si RNA-1 group,si RNA-2 group,si RNA-3 group,NC-si RNA group and control group.Specific si RNA of FGF1 were transfected into 5 groups of cells respectively.The protein level of FGF1 in A549 cells was detected by Western Blot.Selecting si RNA-1,which significantly reduced FGF1 expression,was used as a follow-up tool.2.In the subsequent experiments,the A549 cells were divided into 3 groups(control group,NC-si RNA group,si RNA-1 group).The protein and m RNA levels of MMP-9,E-cadherin,and N-cadherin in three groups of cells were detected by Western Blot and q RT-PCR experiments.3.The changes of mitochondrial membrane potential in three groups of cells were detected by the flow cytometry,thus reflecting the level of apoptosis.4.The Transwell assay was used to determine the invasive ability of three groups of cells.Results:1.After silencing FGF1 gene in A549 cells with si RNA for 48 hours,the protein level of FGF1 in si RNA-1 group and si RNA-2 group was significantly lower than that in the control group(P<0.001,P<0.01,n=6);The protein level of FGF1 in si RNA-3 and NC-si RNA group cells was not significantly different from the control group(P>0.05,n=6).2.The protein and m RNA levels of MMP-9 in the si RNA-1 group were significantly lower than those in the control group(P<0.001,n=6),and the protein and m RNA levels of E-cadherin were significantly higher than those in the control group(P<0.001,n=6);the protein and m RNA levels of N-cadherin did not show significant differences(P>0.05,n=6).The protein and m RNA levels of MMP-9,E-cadherin and N-cadherin in NC-si RNA group had no significant difference compared with the control group(P>0.05,n=6).3.The flow cytometry observed that compared with the control group,the mitochondrial membrane potential of the si RNA-1 group was significantly reduced(P<0.001,n=6),indicating that the proportion of apoptosis was increased.There was no significant difference in mitochondrial membrane potential of NC-si RNA group compared with control group(P>0.05,n=6).4.The Transwell assay showed that compared with the control group,the number of cells entering the lower chamber in the si RNA-1 group was significantly decreased(P<0.001,n=6),reflecting the invasive ability of cells decreased;however,there was no significant change in the NC-si RNA group compared with the control group(P>0.05,n=6).Conclusions: In NSCLC cells,after silencing FGF1 gene by si RNA,the expression of FGF1 protein was decreased,leading to the decreased expression of MMP-9,the increased expression of E-cadherin and the increased proportion of cell apoptosis,ultimately the cell’s invasive ability is reduced.It is suggested that the FGF1 can be a potential target for future treatment of NSCLC patients.