The Inhibition Of Autophagy Of Lung Adenocarcinoma Cell By Rac3 Through MTOR Signal Way

Objective: The morbidity and mortality rates of lung cancer is increasing worldwide.The characteristic of lung cancer is later diagnosis and short survival.Although,radical operation and target therapy can improve disease-free survival and 5 years survival,but the improving is not obvious.Rac3 can modulate cancer cell apoptosis,cell cycle,invasion and immigration.We have detected the expression of Rac3 in lung adenocarcinoma and observed the effect of Rac3 on lung cancer apoptosis,proliferation and immigration.Autophagy is the main factor in cell program death and modulates the cell renew.It is the focus to discover the mechanism of autophagy.In this research,we observed the effect of Rac3 on lung adenocarcinoma autophagy and discuss the mechanism,the aim was to provide new molecular mark for diagnosis and prognosis evaluation,to provide new target for target therapy.Methods: 1.Western blot and qRT-PCR was used to detect the Rac3 expression in four human lung adenocarcinoma cell lines(A549?H1975?H358?NH-1299).2.LV-Rac3-RNAi was transfected into A549 cell to decrease Rac3 expression and pCMV-Rac3 was transfected into H1299 cell to increase Rac3 expression,immunofluorescence was used to identify the transfection efficiency,Western blot and qRT-PCR were used to detect Rac3 expression post-transfection.3.To observe the effect of Rac3 on autophagy,Rapamycin was used to induce autophagy.LC3-?/LC3-? ratio and p62 expression were used to evaluate autophagy.4.To discuss the mechanism,autophagy-related proteins level were detected with Western blot when Rac3 in H1299 was down-regulated.5.To detect the effect of Rac3 on mTOR pathway,amino acid starvation was used to inhibit Rac3 activating mTOR and amino acid re-supplement was used to observe mTOR pathway protein level to identify the effect of Rac3 on mTOR.Results: 1.Rac3 protein and mRNA were expressed in lung adenocarcinoma cellines.2.Lv-Rac3-RNAi and pCMV-Rac3 were transfected into A549 or H1299 cells respectively to reduce or accelerate Rac3 expression,the transfection efficiency was 90% identified by fluorescence microscope.3.Rapamycin induced autophagy in Rac3 down-regulated A549 cells showed that LC3-?/LC3-?ratio was increased and p62 was decreased in 1?m/ml Rapamycin.This means that low expression Rac3 attenuated Rapamycin induced autophagy.4.Rapamycin induced autophagy in Rac3 up-regulated H1299 cells showed that LC3-?/LC3-?ratio was decreased and p62 was increased in 1?m/ml Rapamycin.This means that high expression Rac3 inhibited Rapamycin induced autophagy.5.Western blot showed that p-mTOR,p-ulk1,p-p70 S6 K,p-S6 expression were increased when Rac3 was up-regulated in H1299 cells and Rac3 may activate autophagy by mTOR phosphorylation.6.Amino acid starvation inhibited Rac3 over-expression induced mTOR pathway phosphorylation and re-supplement of amino acid resumed mTOR pathway phosphorylation partly,this further proofed that Rac3 up-regulation inhibited lung adenocarcinoma autophagy by activating mTOR signal.Conclusion: 1.Rac3 down-regulation attenuated Rapamycin induced lung cancer cell A549 autophagy and Rac3 up-regulaion inhibited Rapamycin induced lung cancer cell H1299 autophagy.2.Rac3 up-regulation inhibited lung adenocarcinoma autophagy by activating mTOR signal.