摘要
目的:观察烯脂酰辅酶A水合酶短链1(enoyl-coenzyme A hydratase,short chain1,ECHS1)对肝癌HepG2细胞增殖的影响。方法:将干扰ECHS1基因的重组质粒pGPU6/GFP/siRNA-ECHS1转染至HepG2细胞,通过嘌呤霉素筛选稳定干扰ECHS1基因表达的HepG2细胞,蛋白质印迹法检测细胞中ECHS1蛋白的表达水平。pGPU6/GFP/siRNA-ECHS1转染HepG2细胞后,CCK-8(cellcountingkit-8)和5-溴脱氧尿嘧啶核苷(5-bromo-2’-deoxyuridine,BrdU)法检测细胞的增殖情况,蛋白质印迹法检测细胞中细胞外信号调节激酶(extracellular signal regulated kinase,ERK)、磷酸化ERK(phosphorylated-ERK,p-ERK)、cyclinD3和cyclinD1蛋白的表达水平。结果:成功建立稳定干扰ECHS1基因表达的HepG2细胞,pGPU6/GFP/siRNA-ECHS1转染后HepG2细胞中ECHS1蛋白的表达水平明显低于空白对照组(未转染质粒的HepG2细胞)和阴性对照组(转染空载体pGPU6的HepG2细胞)(P<0.05)。与阴性对照组比较,pGPU6/GFP/siRNA-ECHS1转染后HepG2细胞的增殖能力受到明显抑制(P<0.05),p-ERK、cyclinD3和cyclinD1蛋白的表达水平均明显降低(P<0.05)。结论:ECHS1可能通过上调p-ERK、cyclinD3和cyclinD1的表达而促进肝癌HepG2细胞的增殖。
Objective： To investigate the effect of ECHS1 （enoyl-coenzyme A hydratase, short chain 1） on the proliferation of hepatocellular carcinoma HepG2 cells. Methods： The recombinant plasmid pGPU6/ GFP/siRNA-ECHS1 for ECHS1 gene interference was transfected into HepG2 cells. The HepG2 cells with stable ECHS1 gene interference were screened by puromycin. The expression level of ECHS1 protein in HepG2 cells was detected by Western blotting. The proliferative ability of HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 was detected by CCK-8 （cell counting kit-8） assay and BrdU （5-bromo- 2＇-deoxyuridine） assay. The expression levels of ERK （extracellular signal regulated kinase）, p-ERK （phosphorylated-ERK）, cyclin D3 and cyclin D1 were detected by Western blotting. Results： The HepG2 cells with stable ECHS1 gene interference were successfully established. The expression level of ECHS1 protein in HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 was significantly lower than that in the blank control cells （HepG2 cells without transfection） and the negative control cells （HepG2 cells transfected with pGPU6 vector） （P 〈 0.05）. As compared with the negative control cells, the proliferative ability was significantly inhibited （P 〈 0.05） and the expression levels of p-ERK, cyclin D3 and cyclin D1 proteins were lower in HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 （P 〈 0.05）. Conclusion： ECHS1 may promote the proliferative ability of HepG2 cells via enhancing the expression levels of p-ERK, cyclin D3 and cyclin D1.
出处
《肿瘤》
CAS
CSCD
北大核心
2013年第5期422-427,共6页
Tumor
关键词
肝肿瘤
细胞增殖
细胞外信号调节激酶
基因
ECHS1
细胞
HepG2
Liver neoplasms
Cell proliferation
Extracellular signal-regulated protein kinase
Gene,enoyl-coenzyme A hydratase, short chain 1 Cell, HepG2
