Reversal Of Chemotherapy Resistance To Cisplatin In NSCLC By MiRNA-195-5p Via Targeting The FGF2 Gene

Objective:Compared to the expression and biological behavior of miR-195-5p in non-small cell lung cancer cell(NSCLC)line A549 and cisplatin-resistant cell line A549/DDP,the mechanism of miR-195-5p in the development of NSCLC and cisplatin-resistance was inquired into.Methods:1.Using Rq-PCR,we detected the content of miR-195-5p in A549 and A549/DDP cells and compared the differences in chemotherapy sensitivity,cell invasion and migration ability of the two groups of cells through MTT assay,cell scratch assay and Transwell.The direct effects of miR-195-5p on chemosensitivity,cell invasion and migration of cisplatin-resistant A549/DDP cells were supported by function acquisition and loss experiments.2.Depending on analysis of biological information prediction software—Target Scan Human7.2and miRanda,we identified a possible target gene of miR-195-5p.Luciferase reporter vector was constructed by amplifying FGF2 3’-UTR fragment containing miR-195-5p targeting sites.Expressions of miR-195-5p and FGF2 were checked to test the hypothesis.3.Designing over-expression and lower-expression of FGF2 plasmids,MTT assay,cell scratch assay and Transwell test were also used to test the differential changes in expression of miR-195-5p and FGF2 and cells functions of NSCLC cells.A549/DDP and A549 cells were transfected with overexpressing FGF2 and interfering FGF2 plasmids.MTT assay,cell scratches assay and Transwell were utilized to compare the differences in cell activity,invasion and migration ability between the two groups.4.Determining the expression of EMT-related markers after transfection by western blot.5.Examining the changes on sensitivity of cisplatin in drug resistance cell after transfection through MTT assay.6.Setting up nude mice xenograft model that inoculated A549 or A549 cells transfected with miR-195-5p /miR-195-5p ASO or not,in vivo experiments further confirmed that miR-195-5p affects the chemotherapy-sensitivity and drug resistance of NSCLC patients to cisplatin by inhibiting the FGF2 target gene.Results:1.With reduced chemotherapy sensitivity and increased cell invasion and migration ability,A549/DDP cells have more lower miR-195-5p content than that in A549 cells.The loss-of-function and gain-of-function assays illustrated that miR-195-5p might have increased the chemosensitivity to cisplatin in the A549/DDP cells and decreased cell migration and invasion.2.The differential expressions between miR-195-5p and FGF2,and the dual-luciferase reporter assured that FGF2 was a correlated action target of miR-195-5p negatively.The results of qt-PCR showed that the relative expression level of miR-195-5p in A549/DDP cells was significantly increased,while the expression level of FGF2 was significantly decreased in the miR-195-5p mimics group.3.While FGF2 decreased significantly in FGF2 knockdown cells,the relative expression level of miR-195-5p increased significantly.When the relative expression level of miR-195-5p was reduced in A549 cells,the expression level of FGF2 was also significantly increased.In the cells with high expression of FGF2,FGF2 significantly increased,while the relative expression level of miR-195-5p was also significantly decreased.Changes of FGF2 protein level detected by Western blot were similar to those detected by PCR.It indicates that miR-195-5p has an opposite correlation with FGF2.4.Differential expressions of the EMT-related protein clarified that miR-195-5p might affect EMT by inhibiting FGF2 through western blot.5.Compared the differences in cell activity,invasion and migration ability between the cells transfected with overexpressing FGF2 or interfering FGF2 plasmids,we found that over-expression of FGF2 can inhibit the biological activity,migration and invasion of tumor cells.Analyzing the chemotherapy-sensitivity of two transfection groups to cisplatin through MTT assay,we thought that over-expression of FGF2 resulted in enhanced cisplatin resistance in the cells,while miR-195-5p might have reversed the resistance.6.Tumorigenic A549/DDP cells were larger than the A549 cells,and the expressions of miR-195-5p and FGF2 were correlated negatively.Conclusion:MiR-195-5p might target FGF2 to reverse cisplatin resistance in A549/DDP cells and enhance chemosensitivity,cell invasion and migration ability of the two groups of cells.