The Mechanism Of MGST1 Inhibits Apoptosis Of Human Lung Adenocarcinoma Cell

Background:Lung cancer is one of the most common malignancies,its morbidity and mortality in our country and the world are the highest.Lung adenocarcinoma is one of the most common type of lung cancer.The diagnosis is often in the late stage and the prognosis is poor.Although in recent years the researches in the molecular mechanisms have made great progress,but its pathogenesis is far from been elucidated.Therefore,it is of great significance to study the pathogenic genes of lung adenocarcinoma and to explore the new therapeutic target gene of lung adenocarcinoma.The study shows that MGST1 is closely involved in the development and progression of esophageal cancer,epithelial ovarian cancer and prostate cancer,and it is highly expressed in lung cancer of transgenic mice,but its function and mechanism in lung cancer have not been reported.Our previous studies have shown that MGST1 is highly expressed in human lung adenocarcinoma tissue and inhibits the expression of MGST1 in lung adenocarcinoma cell A549,which inhibits cell proliferation and promotes the apoptosis,but the mechanism has not been elucidated.As MGST1 is expected to become a new candidate target gene for lung adenocarcinoma,further elucidating the mechanism of MGST1 promoting the apoptosis of lung adenocarcinoma will accomplish the goal.Objective:In this study,the expression of endogenous MGST1 in lung adenocarcinoma cell line PC-9 was disrupted by lentivirus and the expression of exogenous MGST1 in lung adenocarcinoma cell line SPC-A-1 was transfected by overexpressed plasmid.The goal is to explore the mechanism of MGST1 in order to obtain a reliable experimental evidence that MGST1 plays an important role in the malignant process of lung adenocarcinoma.It will provide scientific evidence for developing the treatment based on the new candidate gene MGST1 for lung adenocarcinoma.Methods:The expression of MGST1 protein in lung epithelial cell line BEAS-2B and lung adenocarcinoma cell lines SPC-A-1,H2342,A549,H1975,PC-9 and XLA-07 were detected by Western-blot.Then PC-9 and SPC-A-1 were chosen as the tool cells in the later study.The expression of MGST1 was detected by Western-blot.The proliferation activity of cell was detected by MTS assay.The clone formation ability was detected by clone formation assay.The apoptosis was detected by flow cytometry.Related apoptotic proteins changes were detected by Western-blot.SPC-A-1 cell line with stable over-expression of MGST1 was established by constructing the recombinant plasmid and transfection.The expression of MGST1 mRNA was detected by Real-time QPCR and protein was detected by Western-blot.The proliferation activity of cell was detected by MTS assay.The clone formation assay was used to detect Cloning ability.Flow cytometry was used to detect the apoptosis induced by H2O2,and the related apoptosis proteins changes were detected by Western-blot.Results:1.The expression of MGST1 protein in lung epithelial cell line and lung adenocarcinoma cell lines? The expression of MGST1 protein in lung adenocarcinoma cell lines were higher than that in lung epithelial cell BEAS-2B,and the expression of MGST1 protein was significantly increased in H1975,PC-9 and XLA-07 cell lines.The expression in SPC-A-1,H2342,A549 cells was relatively lower.According to the basic expression,PC-9 was used as a cell line for lentivirus-mediated MGST1 interference,SPC-A-1 was choosed to construct the MGST1 stable over-expression cell line.2.Lentivirus disrupts the expression of MGST1 in PC-9 cell and promotes the apoptosis of PC-9 cell? The result of Western-blot showed that the expression of MGST1 protein was significantly decreased,indicating that lentivirus infection succeeded in interfering the expression of target protein.? The result of MTS showed that the proliferation activity of PC-9 cell was significantly inhibited with the interfered MGST1 expression.? The result of clonal formation showed that the cell cloning ability of PC-9 cell was inhibited with the interfered MGST1 expression.? The apoptosis of PC-9 cell detected by flow cytometry was significantly increased with the interfered MGST1 expression.? The expression of cleaved-caspase 9,cleaved-caspase 3 and cleaved-PARP were significantly increased and the expression of caspase 9,caspase 3 and PARP were decreased with the interfered MGST1 expression,which were detected by Western-blot.3.After over-expression of MGST1 gene in SPC-A-1 cell,H2O2-induced apoptosis of SPC-A-1 cell was inhibited? Overexpression plasmids of MGST1 was successfully constructed determined by sequencing.Real-time QPCR and Western-blot results showed that the expression of MGST1 mRNA and protein in SPC-A-1 was increased,indicating that the stable expression of MGST1 SPC-A-1 cell line was successfully established.? The result of MTS showed that the proliferation activity of SPC-A-1 cell was significantly increased after overexpression of MGST1 gene.? The result of clonal formation showed that the clone formation ability of SPC-A-1 cell was significantly enhanced after stable overexpression of MGST1 gene.? Flow cytometry showed that the apoptosis of SPC-A-1 cell induced by H2O2 was significantly decreased after stable overexpression of MGST1 gene.? The expression of caspase 9,caspase 3 and PARP in SPC-A-1 cell induced by H2O2-induced apoptosis of SPC-A-1 cell with overexpressed MGST1 gene was significantly higher than that of the control group,and the expression of cleaved-caspase 9,cleaved-caspase 3,cleaved-PARP expression was significantly reduced.CONCLUSION:Through this study,we found that MGST1 can inhibit the apoptosis of lung adenocarcinoma cells by inhibiting the caspase apoptosis protein 9,3 and PARP.