Roles And Underlying Mechanisms Of MFN2 In Cellular Survivaland Proliferation Of Lung Adenocarcinoma

Objective Platinum-based chemotherapy is the first-line treatment in advanced lung adenocarcinoma.Unfortunately,the research on profiling of gene expression in predicting the efficiency of this treatment has not got exciting results.Our previous study suggested that gene mitofusin2(MFN2)controlled of apoptosis of mitochondrial pathway via MAPK signal transduction pathway,which had the similar pathway on tumor cell apoptosis induced by cisplatin.So we supposed that it might be in synergistic effects in apoptotic mechanisms with MFN2 and cisplatin.In the following study we planned to explore the critical questions:(1)Expression status of MFN2 in lung adenocarcinoma specimens.(2)Roles of MFN2 in the survival,proliferation and invasion of lung adenocarcinoma cells?(3)Underlying mechanisms for the regulation of lung adenocarcinoma survival and proliferation by MFN2?Methods(1)Immunohistochemistry analysis of MFN2 protein expression in 30 pairs of lung adenocarcinoma and adjacent non-tumor normal tissues.(2)Lentiviral-mediated RNAi system for MFN2 knockdown in lung adenocarcinoma cell lines to investigate the impact of MFN2 knockdown on cellular survival,proliferation and invasion with MTT assay,cell cycle analysis,cell apoptosis and cell invasion assay.(3)impact of MFN2 knockdown on lung adenocarcinoma proliferation in vivo by xenograft lung adenocarcinoma model.(4)microarray analysis to identify downstream targets of MFN2 for the regulatory network analysis of differential genes.(5)high-throughput screening to identify downstreamtargets of MFN2 involved in lung adenocarcinoma proliferation.(6)expression status of selected targets in lung adenocarcinoma specimens.(7)lentiviral-mediated RNAi system to inhibit selected targets for functional analysis in lung adenocarcinoma.Results(1)Immunohistochemistry analysis showed that MFN2 expression was significantly higher in lung adenocarcinoma specimens as compared to adjacent non-tumor normal tissues.(2)lentiviral-mediated RNAi system inhibited the expression of MFN2 at both RNA and protein levels efficiently.Functional analysis revealed that MFN2 knockdown inhibited cellular proliferation,cell cycle process and cell invasion in lung adenocarcinoma cells A549.(3)xenograft lung adenocarcinoma model showed that MFN2 knockdown inhibited the tumorigeneses of lung adenocarcinoma cells A549 in vivo.(4)microarray analysis showed that pathways invovled in cell cycle process was inhibited in lung adenocarcinoma cells A549 with MFN2 knockdown.It was shown that RAP1 A,RALB and ITGA2 were differentially regulated by MFN2,which indicated that MFN2 regulated tumorigenesis in lung adenocarcinoma bi-directionally.(5)high-throughput screening identified MLF1 IP was one of the downstream targets of MFN2 and involved in cell proliferation regulation in lung adenocarcinoma.(6)MLF1IP expression was significantly higher in lung adenocarcinoma speciemens as compared to adjacent non-tumor normal tissues.(7)lentiviral-mediated RNAi system inhibited MLF1 IP efficiently and MLF1 IP knockdown inhibited the proliferation,colony formation ability and cell cycle process while promoted cell apoptosis in lung adenocarcinoma cells A549.Conlusion(1)MFN2 is critical for lung adenocarcinoma cell survival,proliferation and invasion ability.MFN2 knockdown promotes lung adenocarcinoma apoptosis.(2)MFN2 regulated lung adenocarcinoma survival and proliferation via downstream target MLF1 IP.