Effect And Mechanism Of MiRNA-200a Targeting Regulation Of HGF/c-metexpression On Radiosensitivity Of Non-small Celllung Cancer

【Objective】To investigate the biological function and molecular mechanism of miRNA-200 a by inhibiting the HGF / c-met pathway in non-small cell lung cancer(NSCLC)with ionizing radiation,and to provide new biomarkers and new molecular therapy target for clinical radiotherapy of NSCLC.【Methods】Part Ⅰ:1.The expression of HGF in lung cancer tissues and adjacent normal tissues was detected by immunohistochemistry and Western Blot.2.Using real-time fluorescence quantitative PCR and Western Blot assays,we examined the effect of HGF siRNA on the expression of HGF in NSCLC cells.3.Transwell test was used to detect the migration and invasion of NSCLC cells with low expression of HGF.Part Ⅱ: 1.After screening of microRNAs corresponding to HGF as target genes,real-time quantitative PCR was used to detect the expression of microRNAs in lung cancer and corresponding normal tissues.2.The expression of HGF in NSCLC cells was detected by real-time fluorescence quantitative PCR.3.The fluorescence in situ hybridization was used to detect the expression of miRNA-200 a in lung cancer and corresponding normal tissues.4.The dual luciferase reporter gene system was used to verify whether miRNA-200 a inhibited the 3’-UTR of HGF.5.The expression of HGF and c-met protein in overexpressing miRNA-200 a NSCLC cells was detected by Western Blot assays.6.Using transwell assay to test the migration and invasion of overexpressing miR-200 a NSCLC cells.Part Ⅲ: 1.The apoptotic changes of NSCLC cells with low expression of HGF and overexpression of miRNA-200 a were detected by flow cytometry.2.Using immunofluorescence staining to detect the changes of DNA damage induced by ionizing radiation in NSCLC cells with of HGF and overexpression of miRNA-200 a,respectively.3.The radiosensitivity of NSCLC cells of low expression of HGF and overexpressing miR-200 a was detected by clonal assay at different ionizing radiation dose.【Results】Part Ⅰ: 1.The expression of HGF in lung cancer and the corresponding normal lung tissues was significantly different,and it was significantly increased in lung cancer tissues.2.HGF siRNA can significantly inhibit the expression of HGF and c-met in NSCLC cells.3.Low expression of HGF can reduce the migration and invasion of NSCLC cells.Part Ⅱ: 1.MiRNA-200 a and miRNA-141 were screened out with HGF as target gene,and miRNA-200 a decreased significantly in lung cancer tissues.2.After transfection of microRNA mimics,the expression of HGF in NSCLC cells of upregulated miRNA-200 a were decreased significantly.3.The expression of miRNA-200 a in lung cancer tissues was significantly lower than that in normal lung tissues.4.miRNA-200 a can bind to the 3’-UTR of target gene HGF,thus play a role in inhibiting HGF.5.After overexpression of miRNA-200 a,the expression of HGF and c-met protein in NSCLC cells decreased.6.After upregulated miRNA-200 a,the migration and invasion of NSCLC cells were reduced.Part Ⅲ: 1.The apoptosis rate of NSCLC cells with low expression of HGF or overexpressing miRNA-200 a was significantly raised.2.The DNA damage of NSCLC cells with low expression of HGF or overexpression of mi RNA-200 a was significantly increased compared with that in the normal group after ionizing radiation.3.The survival fraction of NSCLC cells with low expression of HGF or overexpressing miRNA-200 a was significantly decreased and the radiosensitivity was enhanced under the irradiation of 0Gy、2Gy、 4Gy、 6Gy and 8Gy doses.【Conclusion】In the progression of NSCLC,the expression level of miRNA-200 a was decreased,the inhibitory effect of miRNA on the regulation of target gene HGF was reduced,the expression level of target gene HGF was increased,and the c-met signal pathway was further activated,which accelerated the radiation resistance and resulted in the progress of NSCLC.