Significance Of MGST1 Expression In Lung Adenocarcinoma And Its Mechanism Of Function On Lung Adenocarcinoma Cells

Objectives:To analyze the expression level of MGST1 in lung adenocarcinoma and its clinical significance;to study the effects of MGST1 knockdown on the biological functions of lung adenocarcinoma cell proliferation,apoptosis,invasion,migration and growth ability in nude mice;to investigate preliminary mechanisms of biological function of MGST1 in lung adenocarcinoma.Methods:1.Analysis of MGST1 differential expression in lung cancer tissue and normal lung tissue which were attained from GEO and TCGA database were performed;IHC technology was used to detect the expression level of MGST1 in lung adenocarcinoma tissue;Chi-square test was used to analyze the relationship between the expression level of MGST1 and clinical pathological parameters of patients with lung adenocarcinoma;Kaplan-Meier plotter prediction website and survival curve were used to analyze the effect of MGST1 expression on the prognosis of patients with lung adenocarcinoma.2.qRT-PCR and Western blot were used to detect the expression level of MGST1 in lung adenocarcinoma cell lines;MGST1 knockdown lentiviral(MGST1 shRNA)was constructed and used to infect two lung adenocarcinoma cell lines with MGST1 high expression;qRT-PCR and Western blot were used to detect the knockdown efficiency of MGST1 shRNA;MTS,EdU and plate cloning experiments were used to detect the effect of MGST1 shRNA on cell proliferation;flow cytometry was used to detect the effect of MGST1 shRNA on cell apoptosis;Western blot was used to detect the effect of MGST1 shRNA on apoptosis-related protein of caspase family and Bcl-2 expression;Boyden chamber test was used to detect the effect of MGST1 shRNA on cell invasion ability;Transwell chamber test was used to detect the effect of MGST1 shRNA on cell migration ability;the effect of MGST1 shRNA on the growth capacity of lung adenocarcinoma cells in nude mice was measured by subcutaneous tumor formation experiments.3.Full transcriptome sequencing technology was used to detect differentially expressed genes in A549 cells before and after MGST1 knockdown,and biological information websites was used to analyze the relationship between differentially expressed genes and biological functions of cancer,and classic signaling pathways;Western blot was used to detect the effect of MGST1 shRNA on AKT/GSK3?/?-catenin signaling pathway;AKT activator SC79 was used to perform functional recovery experiments.Functional experiments were used to detect the effect of MGST1 on proliferation,and migration ability of lung adenocarcinoma cells by regulating the AKT/GSK3?/?-catenin signaling pathway.Results:1.GEO database analysis showed that the expression of MGST1 in lung cancer tissues was higher than that in normal lung tissues(p<0.05);TCGA database analysis showed that MGST1 expression in lung adenocarcinoma tissues was higher than that in normal lung tissues(p<0.05);the positive expression rate of MGST1 in 173 lung adenocarcinoma tissues(76.30%)was higher than that in its matched distal non-cancer tissues(41.62%)detected by IHC(p<0.05);Positive expression of MGST1 in lung adenocarcinoma tissues was positively correlated with patients' AJCC stage(p<0.05);patients with positive MGST1 expression had poor prognosis and MGST1 was an independent risk factor for poor prognosis in patients with lung adenocarcinoma.2.Compared with the normal bronchial epithelial cell line BEAS-2B,the expression of MGST1 was increased in lung adenocarcinoma cell lines NCI-H2342,NCI-H1975,A549 and PC-9,and was higher in A549 and PC-9 cells(p<0.05);the constructed MGST1 shRNA significantly reduced the expression of MGST1 mRNA and protein in lung adenocarcinoma cells A549 and PC-9 cells(p<0.05);MTS,EdU and plate clone experiments showed that compared with the scramble group,proliferation ability of A549 and PC-9 cells in shMGST1-1 and shMGST1-2 groups were reduced(p<0.05);flow cytometry results showed that compared with the scramble group,early stage apoptosis rate of A549 cells in shMGST1-1 and shMGST1-2 groups were increased(p<0.05);compared with the scramble group,the apoptosis-related proteins cleaved-caspase9,cleaved-caspase3,and cleaved-PARP expression of A549 cells in the shMGST1-1 and shMGST1-2 groups were increased and anti-apoptotic protein Bcl-2 expression was decreased(p<0.05);Boyden chamber test results showed that compared with the scramble group,the invasion ability of A549 and PC-9 cells in the shMGST1-1 and shMGST1-2 groups were reduced(p<0.05);Transwell chamber test results showed that compared with the scramble group,cell migration ability of A549 and PC-9 cells in shMGST1-1 and shMGST1-2 groups were reduced(p<0.05);the results of subcutaneous tumor formation experiments in nude mice showed that compared with the scramble group,the growth rate,tumor formation volume and tumor weight of A549 cells in shMGST1-1 group were all reduced(p<0.05).3.Whole transcriptome sequencing technology and bioinformatics analysis showed that MGST1 regulates 10042 differentially expressed genes in A549 cells,of which 4899 genes were up-regulated after MGST1 knockdown and 5143 genes were down-regulated;Functional analysis(GO Analysis)showed that differentially expressed genes were associated with cell growth,apoptosis,and invasion;Cell pathway analysis showed that differentially expressed genes were enriched in the AKT signaling pathway;Western blot results confirm that knockdown of MGST1 inhibited expression of key proteins pAKT,pG3K-3? and ?-catenin in the AKT/G3K-3?/?-catenin signaling pathway(p<0.05);Functional tests showed that AKT activator SC79 attenuated the inhibitory effects of MGST1 shRNA on cell proliferation and migration ability of lung adenocarcinoma cells(p<0.05).Conclusions:1.MGST1 is highly expressed in lung adenocarcinoma tissues and cell lines.2.The high expression of MGST1 is positively correlated with the AJCC stage of patients,and it is a molecular marker of poor prognosis of patients with lung adenocarcinoma.3.Knockdown of MGST1 expression inhibits lung adenocarcinoma cell proliferation,invasion,migration ability and growth ability in nude mice,and promotes cell apoptosis.MGST1 may be a candidate target for lung adenocarcinoma treatment.4.MGST1 may promote the malignant progression of lung adenocarcinoma by regulating the AKT/GSK3?/?-catenin signaling pathway.