靶向干扰CXCR7基因表达对肝癌(HCC)细胞增殖及凋亡的影响

Effects of inhibiting CXCR7 gene expression on biological characteristics of human hepatocellular carcinoma(HCC)cell lines

目的探讨CXCR7基因在不同肝癌细胞系及肝癌组织中的表达情况,并研究靶向抑制CXCR7基因表达对肝癌细胞增殖及凋亡的影响。方法应用Western blot检测不同肝癌细胞系及10例肝癌组织中CXCR7蛋白的表达水平。采用短发夹状RNA(short hairpin RNA,shRNA)干扰技术沉默HCCLM3与MHCC97L细胞中CXCR7基因的表达,分别采用Western Blot和qRT-PCR检测shRNA的靶向沉默效果。通过CCK-8增殖实验与Annexin V-FITC/PI细胞双染色研究CXCR7抑制对HCCLM3与MHCC97L增殖及凋亡的影响。结果CXCR7表达水平与肝癌细胞的恶性程度呈正相关,肝癌组织中CXCR7表达水平较癌旁显著升高。实验成功构建了9个慢病毒表达载体,其中CXCR7shRNA-566序列干扰效率最高。增殖实验显示干扰表达组细胞生长速度受到明显抑制(P<0.05);流式细胞仪分析显示干扰CXCR7表达可诱导细胞凋亡的发生。结论 CXCR7表达水平与肝癌恶性程度呈正相关,靶向干扰CXCR7表达可抑制细胞的增殖能力,诱导细胞发生凋亡。

Objective To explore the expression of chemokine receptor CXCR7 on human hepatocellular carcinoma （HCC） cell lines and tumor samples,and the effects of CXCR7 on cell proliferation and apoptosis of HCC cell lines.Methods Western blot analysis was applied to detect CXCR7 protein expression in HCC cell lines and 10 pairs of HCC specimens with adjacent tissues.RNA interference method of short hairpin RNA （shRNA） was used to silence CXCR7 expression in HCCLM3 and MHCC97L cells.The expressions of CXCR7 mRNA and protein were determined by qRT-PCR and Western blot,respectively.The effects of CXCR7 down-regulation on cell proliferation and apoptosis were measured by CCK-8 assay and Annexin V-FITC/PI apoptosis detection.Results The CXCR7 expression was gradually enhanced with increasing metastatic potential of HCC cell lines,and compared to adjacent tissues of clinical specimens,CXCR7 expression was hugely increased in tumor tissues.Nine lentiviral expressing vectors were constructed successfully and stably transfected into HCCLM3 cells.Based on their effects on CXCR7 expression,CXCR7 shRNA566 was selected to evaluate the effects of down-regulation of CXCR7 on proliferation and apoptosis of HCCLM3 and MHCC97L cells in vitro.It was found that CXCR7 shRNA566 could significantly inhibit the proliferation of HCCLM3 and MHCC97L cells （P〈0.05）.Flow cytometry assay showed that depletion of CXCR7 expression could induce cell apoptosis.Conclusions CXCR7 expression is positively associated with HCC progression,and interference targeted of CXCR7 expression inhibit cell proliferation and induce apoptosis.