RNA干扰CXCR7基因对人肝癌MHCC97H细胞生物学特性的影响

Effects of Down-regulating CXCR7 Gene Expression on Biological Characteristics of Human Hepatocellular Carcinoma Cell Line MHCC97H

通过构建针对CXCR7的短发夹状RNA(short hairpin RNA,shRNA)干扰表达质粒,转染人肝癌细胞系MHCC97H,嘌呤霉素筛选建立低表达CXCR7基因的稳转株,分别采用qRT-PCR和Western Blot检测shRNA靶向沉默效果。通过CCK-8增殖实验与流式细胞术研究CXCR7抑制对细胞增殖及凋亡的影响;通过划痕愈合实验研究CXCR7抑制对MHCC97H细胞迁移能力的影响。结果表明:成功构建了慢病毒表达载体pLKO.1-CXCR7shRNA,显著下调MHCC97H细胞中CXCR7mRNA及蛋白的表达。增殖实验显示,与阴性对照组相比,干扰表达组细胞生长速度受到明显抑制(p<0.001);干扰CXCR7表达可诱导细胞凋亡,尤以早期凋亡率上调为著;划痕实验显示抑制CXCR7表达延长细胞的伤痕愈合时间。

Lentiviral expressing vectors of small hairpin RNA aimed at CXCR7 gene were constructed and transfected into MHCC97H cell line. Cell lines which stably expressed low level of CXCR7 were estab lished by puromycin screening. The mRNA and protein expression of CXCR7 were measured by RT-PCR and western blot analysis, respectively. The effects of down-regulation of CXCR7 on cell proliferation, ap-optosis and migration of MHCC97H cell line were evaluated by CCK-8 assay, flow cytometry assay and wound scratch assay, respectively. The results showed that lentiviral expressing vectors were constructed successfully and stably transfected into MHCC97H cells which could dramatically reduce the expressions of CXCR7 both in the level of mRNA and protein. CCK-8 assay showed that shRNA targeting CXCR7 gene could hugely inhibit cell proliferation of MHCC97H in comparison with those in the control groups （p〈 0. 001）. Down-regulation of CXCR7 expression could induce cell apoptosis, especially the early phase ap ootosis. Wound scratch assay also indicated that CXCR7 depletion could 19rolong wound healing hours.