摘要
目的观察趋化因子受体CCR1在高转移潜能人肝癌细胞HCCLM3侵袭中的作用。方法设计并合成3对靶向CCR1 mRNA的寡核苷酸片段,将其连接pcDNA6.2-GW/EmGFP-miR载体构建表达microRNA(miRNA)真核载体S1、S2、S3,脂质体转染法将重组质粒转入HCCLM3细胞。实时荧光定量聚合酶链式反应(real-time PCR)分析CCR1 mRNA抑制效率;Western印迹测定CCR1蛋白的表达量改变;MTT法检测HCCLM3增殖的影响;Matrigel侵袭实验检测HCCLM3细胞侵袭力的改变。结果转染48 h后,S3组CCR1 mRNA较空转染组(Mock)和非特异序列组(Neg)减少80%以上,且明显少于其他组(P<0.01);72 h后S3组CCR1蛋白表达明显少于其他组;转染后,HCCLM3细胞的增殖改变差异无统计学意义。Matrigel侵袭实验结果示转染S3后HCCLM3细胞穿过人工基底膜的数量减少80%,显著低于其他组(P<0.01)。结论人肝癌细胞HCCLM3的CCR1表达可被质粒导入的外源性miRNA有效抑制,进而降低HCCLM3细胞侵袭性,但对细胞增殖无明显影响。
Objective To investigate the role of CCR1 in the invasion of human hepatoeellular carcinoma （HCC） cell line by introduction of exogenous miRNA to silence CCR1 gene expression. Methods Three double-stranded DNA peDNA6.2-GW/ EmGFP-miR vectors （S1, S2, S3 ） targeting human CCR1 mRNA and a negative control plasmid （Neg） were constructed, and were transfeeted into human HCCLM3 cells with high metastatic potentials. Real-time PCR and Western blotting were used to quantify the mRNA and protein levels of CCR1, respectively. Proliferation and invasiveness of transfeeted HCCLM3 cells were analyzed. Results After transfeetion, CCR1 mRNA and protein levels of S3 were significantly lower than the other groups. There was no significant difference in cellular growth rate among the 5 groups. The migrating number of HCCLM3 cells transfeeted with S3 was also significantly decreased as compared with other groups. Conclusion Introduction of exogenous miRNA to HCCLM3 cell line by transfeeting with S3 can effectively reduce the CCR1 expression, which can decease the invasiveness of HCCLM3, but had no significant effect on the cellular proliferation.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第7期784-786,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30471668)
