The Study On The Effect And Molecular Mechanism Of BCAT1 Regulating Gastric Cancer Cell Metastasis

Background:Gastric cancer(GC)is a common malignant tumor of digestive tract in China According to the cancer statistics from International Agency for Research on Cancer in 2018,the morbidity of gastric cancer is ranked fifth and its mortality rate ranks third in 185 countries worldwide.However,in China,both morbidity and mortality are second,and the morbidity in rural areas is higher than that in cities,which brings great challenges to the health of residents,The diagnosis and treatment of gastric cancer has become an urgent public health problem.Big clinical data show that metastasis is the main cause of death in patients with gastric cancer.Further analysis of the mechanism of disease in molecular terms is helpful to improve the therapeutic effect of metastatic gastric cancer Metabolic reprogramming has been considered to be an important feature of tumors,and metabolic abnormalities in tumors have gradually become one of the research hotspots.Branched-chain amino acids(BCAAs)and its metabolic enzymes(Branched-chain amino acid transaminase,BCAT)have been shown to be associated with cell growth and survival in various tumors.Our previous metabolomic prolifing of gastric cancer tissues showed that the levels of BCAAs were significantly increased in gastric cancer tissues,but the role of BCAAs metabolism and its metabolic enzyme BCAT1 in the development of gastric cancer is not yet clear.Therefore,this study focuses on the role and the related mechanisms of BCAT1 in the metastasis of gastric cancer.We will conduct research through the following methods:Firstly,we analyzed the transcription level of BCAT1 in gastric cancer tissues based on the TCGA database and analyze the relationship between BCAT1 expression and patient prognosis.Then verified the differential expression of BCAT1 between clinical gastric cancer tissue samples and normal controls by Western blot,Q-PCR and immunohistochemistry.Next,gastric cancer cell lines with high and low expression of BCAT1 were screened.According to results,we used siRNA to transiently knock down the expression of BCAT1 in MGC803 and then performed the Transwell assay to detect the cell proliferation and migration ability.In order to further verify the role of BCAT1 in the growth of gastric cancer cells,we constructed the monoclonal cell lines with stable knockdown or overexpression of BCAT1 using lentivirus.The migration and invasion ability were detected by Transwell tests and scratch tests,respectively.Then,we explored the molecular mechanism of BCAT1 affecting cell growth changes by verifying the markers of EMT.Finally,we performed proteomic analysis based on the monoclonal cell line that stably knocked down BCAT1 to explore changes in cellular molecular signaling pathways and related proteinsWe have the following results:The expression of BCAT1 significantly increased in gastric cancer tissues based on the database analysis and clinical sample experiments,and the survival time of patients with high BCAT1 expression is significantly shorter than those with lower BCAT1 expression.According to results,the BCAT1 was up-expressed in MGC-803 and down-expressed in AGS.After transient transfection of siRNA to MGC-803 and knocking down BCAT1,the cell migration ability was significantly reduced.The stably low expression or over expression of BCAT1 monoclonal cell lines were successfully construct via lentivirus infection,which can be used in subsequent experiments.After stably knocking down BCAT1,the cell migration and invasion ability was significantly reduced in vitro.After overexpressing BCAT1 in AGS,cell migration and invasion were obviously promoted.The results of RT-PCR and Western blot showed that,t the interstitial markers N-cadherin and Vimentin were significantly decreased,and the cell epithelial markers E-cadherin was notably increasedafter knocking down BCAT1.On the other hand,after overexpression BCAT1,the expression levels of interstitial markers and epithelial markers were reversed.The results combining with proteomics and Western blot suggested that the activation of the Akt/?-catenin signaling pathway was inhibited after knocking out BCAT1,resulting in decreased expression of ?-catenin into the nucleus,thereby inhibiting cell migration and invasion.We come to the following conclusion:The expression level of BCAT1 in gastric cancer tissues was significantly higher than that in normal control tissues,and its expression level was significantly correlated with patient survival.After knocking down BCAT1,the ability of cell migration and invasion is inhibited.While overexpression of BCAT1 can promote cell migration and invasion.Of note,knocking down BCAT1 in the cells inhibit the activation of Akt/?-catenin signaling pathway leads to a decrease in the expression of ?-catenin into the nucleus,which leads to a decrease in cell migration and invasion.Through the above research,we have further clarified the molecular mechanism of BCAAs metabolism regulated by BCAT1 in the metastasis of gastric cancer cells,providing new targets for the future personalized medical treatment of gastric cancer and the development of small molecule inhibitors.