DARS2 promotes EMT,migration and invasion of bladder cancer through regulating EGFR
Objective To investigate the role of mitochondrial aspartyl-TRNA synthetase 2(DARS2)in epithelial mesenchymal trans-formation(EMT),migration and invasion of bladder cancer cells,and explore its underlying mechanism.Methods DARS2 expression in bladder urothelial carcinoma(BLCA)and its relationship with clinicopathological stage were analyzed using TIMER and GEPIA data-bases.RT-qPCR was used to measure the DARS2 mRNA expression in human normal bladder epithelial cells SV-HUC-1 and bladder cancer cells 5637,T24 and TCCSUP.TCCSUP cells were divided into control group(no transfection),si-NC group(transfected with negative control siRNA),si-DARS2-1 group(transfected with DARS2 siRNA-1),si-DARS2 group(transfected with DARS2 siRNA-2),si-DARS2+Vector group(co-transfected with DARS2 siRNA-2 and empty plasmid)and si-DARS2+OE-EGFR group(co-transfected with DARS2 siRNA-2 and EGFR overexpressing plasmid).Western blot was used to detect the expression of EMT markers(E-cadherin,N-cadherin,Vimentin)and EGFR.Transwell invasion assay and cell scratch assay were conducted to detect cell invasion and migration ability,respectively.Results DARS2 mRNA level was significantly higher in BLCA tissue than in adjacent normal tissue,and it was positively correlated with the pathological stage of the disease(F=3.57,P=0.028 9).Compared with immortalized human normal bladder epithelial SV-HUC-1 cells,DARS2 expression was significantly increased in bladder cancer cells 5637,T24 and TCCSUP(P<0.05).Compared to si-NC group,the protein expression levels of N-cadherin,Vimentin,and EGFR were significantly downregulated in si-DARS2 group(P<0.05),while the protein expression of E-cadherin was significantly upregulated(P<0.05).The number of cell invasion and the cell mobility were significantly lower in si-DARS2 group than in si-NC group(P<0.01).Compared with si-DARS2+Vector group,the expression of E-cadherin was significantly decreased in si-DARS2+OE-EGFR group(P<0.05),while the expressions of N-cadherin and Vimentin were significantly increased(P<0.05).Compared with si-DARS2+Vector group,the number of cell invasion and the cell mobility in si-DARS2+OE-EGFR group were significantly increased(P<0.01).Conclusion DARS2 knockdown can inhibit bladder cancer EMT,invasion and migration via downregulating EGFR expression.