BCAT1 Overexpression Induces Metabolic Reprogramming To Promote Lung Adenocarcinoma Cells Proliferation,Migration And Invasion

BackgroundLung cancer is one of the most malignant tumors,among which non-small cell lung cancer(NSCLC)accounts for about 85%.NSCLC mainly consisted of lung adenocarcinoma(LUAD)and squamous cell carcinoma(SCC).Among NSCLC cases,LUAD patients account for about 60%.The recurrence rate of LUAD is high while its 5-year overall survival rate is very low,which is the main cause of lung cancer mortality.Therefore,it is very important to find key factors and study their molecular mechanisms during LUAD development,which contibutes to clinical prevention and treatment of LUAD.Branched-chain amino acid transaminase 1(BCAT1)is associated with a variety of human malignancies.Studies have shown that BCAT1 was highly expressed in various tumor tissues such as gastric cancer?colorectal cancer?prostate cancer?breast cancer?ovarian cancer?liver cancer,and so on.High expression level of BCAT1 was closely related to the poor prognosis of various cancers,as well as the proliferation?migration and invasion abilities of tumor cells.Here we detected expression level of BCAT1 in multiple clinical LUAD samples,and found that BCAT1 was over-expressed in cancer tissues,indicating that BCAT1 was probably involved in the development of LUAD.Therefore,this research is aimed to study the mechanism of BCAT1 in LUAD development,and provide experimental evidence for clinical LUAD diagnosis and treatment.PurposeThe research aims to study the function and molecular mechanism of BCAT1 on the development of lung adenocarcinoma in vitro.Methods1.A tissue microarray containing 93 human LUAD tissues and 85 paracancerous tissues was used to detect the expression level of BCAT1 protein in lung adenocarcinoma tissues and adjacent tissues by immunohistochemical method.The grade of LUAD depended on the intensity and positive rate of BCAT1 in tissues.2.A549 cells were transfected with lentivirus,and positive clones were selected by puromycin to construct a lung adenocarcinoma cell line stably overexpressing or knocking down BCAT.The cells were collected to extract RNA and protein,and the overexpression and knockdown effects were detected by qPCR and Western blotting.3.Cell proliferation ability was measured with a real-time label-free cell function analyzer,which monitored the cell impedance(depending on the number and shape of attached cells)every 5 minutes for 70 hours.The colony forming test was used to detect the colony forming ability of the cells,and the effect of BCAT1 on the migration and invasion ability of A549 cells was detected by scratch repair and transwell experiments.4.Transcriptome sequencing method was used to identify differentially expressed genes between BCAT1 over-expressed cells and control cells,and the altered signal pathways and biological functions were analyzed by bioinformatics methods.5.Glycolysis and mitochondrial stress tests were performed with the seahorse XF-96 cell bioenergy analyzer,and the real-time extracellular acidification rate(ECAR)represented glycolysis ability while the cell oxygen consumption rate(OCR)represented mitochondrial aerobic respiration ability.6.Free fatty acid assay kit was used to measure the free fatty acid content in the cells,and qPCR and Western blot methods were used to detect lipid and glutamine metabolism-related genes expression levels.Results1.The expression level of BCAT1 in clinical lung adenocarcinoma tissues was significantly higher than that in adjacent tissues.2.BCAT1 over-expression significantly enhanced the proliferation rate and colony-forming ability of LUAD cells,accelerated the speed of scratch healing,and enhanced the number of migrating and invading cells.BCAT1 knockdown inhibited the abilities of proliferation,colony formation,scratch healing,migration and invasion of LUAD cells.3.A total of 155 differentially expressed genes were identified by transcriptomics testing of BCAT1 over-expressed and the control group of LUAD cells.Compared with the control group,96 genes were up-regulated and 59 genes were down-regulated.The differentially expressed genes were involved in energy metabolism pathways such as carbohydrate,lipid and amino acid metabolism.4.BCAT1 over-expression significantly inhibited mitochondrial aerobic respiration in LUAD cells.The expression levels of mitochondrial aerobic respiration-related enzymes:pruvate dehydrogenase(PDH),citrate synthase(CS),cytochrome c,voltage-dependent anion channel(VDAC),succinate dehydrogenase(SDHA)were significantly reduced.BCAT1 over-expression had no significant effect on glycolytic levels in LUAD cells,and the expression levels of two key glycolytic enzymes hexokinase 2(HK2)and lactate dehydrogenase A(LDHA)were not changed.5.BCAT1 over-expression affected lipid synthesis and differentiation in LUAD cells and the transcription levels of associated genes long pentraxin-3(PTX3),arrestin domain-containing protein 4(ARRDC4),and deleted in liver cancer 1(DLC1)were elevated in BCAT1 over-expressed cells.BCAT1 overexpression had little effect on fatty acid decomposition.6.Over-expression of BCAT1 promoted glutamine catabolism in LUAD cells.The expression levels of glutaminase(GLS)and glutamate dehydrogenase(GLUD1)increased,and the expression level of glutamine synthetase(GS)decreased significantly in BCAT1 over-expressed cells.Conclusions1.BCAT1 is highly expressed in lung adenocarcinoma tissues and promotes the proliferation,migration,and invasion of LUAD cells.2.BCAT1 over-expression promotes metabolic reprogramming of LUAD cells by inhibiting mitochondrial aerobic respiration,accelerating glutamine metabolism,lipid synthesis and differentiation.