| Background and purpose:Branched-chain amino acid transferase-2(BCAT2)is a key enzyme in branched-chain amino acid metabolism and plays an important role in lipid metabolism and glycolysis.Previous studies have shown that BCAT2 is aberrantly highly expressed in many tumors and can induce tumor proliferation.However,the role of BCAT2 in esophageal squamous cell carcinoma(ESCC)is not clear.The purpose of this study was to explore the effects of BCAT2 in ESCC,clarify its possible molecular mechanism;and made a contribution in molecularly targeted therapies for ESCC patients targeting BCAT2.Methods:1.Differential expression of BCAT2 in ESCC:the differential expression of BCAT2 in esophageal cancer was analyzed by bioinformatics analysis of online database UALCAN,and the protein and mRNA levels of BCAT2 in esophageal squamous cell carcinoma and human immortalized esophageal epithelial cell Het-1A were detected by Western blot and qRT-PCR.2.Functional analysis of BCAT2 in esophageal squamous cell carcinoma:KYSE30 and Eca109 with relatively high BCAT2 expression were screened and transfected with BCAT2 siRNA or pCMV-Myc-BCAT2 to detect the effects of BCAT2 knockdown and overexpression on ESCC cell proliferation,migration and invasion.The effects of BCAT2 knock-down and overexpression on the expression of glycolysis-related hexokinase 2(HK2),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA)and invasion-related neurocalciferol(N-Cadherin)and epithelial calmodulin(E-Cadherin)proteins were detected by Western blot.3.Detection of iron ions,MDA,GSH content and cell viability in ESCC cells after BCAT2 knockdown and Erastin treatment,and the expression of BCAT2 after Erastin treatment was detected by Western blot.4.Detection of iron ion,MDA,GSH concentration and cell viability in ESCC cells treated with BCAT2 knockdown combined with Erastin treatment and BCAT2 overexpression combined with Erastin treatment,CCK-8 was used to detect the viability of BCAT2 knockdown combined with Fer-1 treatment of ESCC cells.5.The effect of BCAT2 knock-down on the expression of Nrf2 protein were detected by Western blot.Results:1.The online database Ualcan showed that BCAT2 was significantly more expressed in esophageal cancer tissues than in normal tissues;BCAT2 differed in protein expression and mRNA levels between ESCC and Het-1 A cells in Western blot and qRT-PCR results,with significant differences between KYSE30 and Eca109.2.BCAT2 knockdown inhibited ESCC proliferation and invasion,while BCAT2 overexpression significantly promoted proliferation and invasion.Both BCAT2 knockdown and Erastin induced the development of iron death phenotype in ESCC,and Erastin down-regulated BCAT2 protein expression in ESCC.4.BCAT2 knockdown combined with the iron death inducer,Erastin,showed more significant intracellular iron ion accumulation,GSH depletion,increased MDA concentration and decreased proliferation capacity;BCAT2 overexpression combined with Erastin showed a more significant decrease in iron ion accumulation,GSH depletion,MDA concentration and proliferation capacity compared with that of the ESCC.BCAT2 overexpression combined with Erastin treatment showed lower iron ion accumulation,increased GSH content and lower MDA concentration compared with Erastin treatment alone;Fer-1 rescued the low proliferation induced by BCAT2 knockdown.5.BCAT2 positively correlated with iron death inhibitory protein Nrf2 and its bound antioxidant response element,and both Erastin and BCAT2 knockdown downregulated Nrf2 protein expression.Nrf2 protein expression was downregulated by both Erastin and BCAT2 knockdown.Conclusions:1.BCAT2 knockdown significantly impedes the proliferation,migration and invasion of ESCC by inhibiting the expression of glycolysis-related proteins and the EMT process,suggesting that BCAT2 may be a new therapeutic target for ESCC.2.BCAT2 exerts pro-tumor effects by regulating iron death in ESCC,suggesting that inhibition of BCAT2 may be a potential therapeutic target for ferroptosis drugs in ESCC. |
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