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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 chemokine receptor CCR1 in the invasion of HCCLM3 cells with high metastatic potential. Methods Three pairs of oligonucleotide fragments targeting CCR1 mRNA were designed and synthesized. The eukaryotic vectors S1, S2, S3 expressing microRNA (miRNA) were constructed by ligating them with pcDNA6.2-GW / EmGFP-miR vector. Method will be recombinant plasmid into HCCLM3 cells. Real-time PCR analysis of CCR1 mRNA inhibition efficiency; Western blot determination of CCR1 protein expression changes; MTT assay HCCLM3 proliferation; Matrigel invasion assay HCCLM3 cells invasiveness changes. Results After 48 h of transfection, CCR1 mRNA in S3 group was reduced by more than 80% compared with Mock group and Neg group (P <0.01), and CCR1 mRNA in S3 group was significantly lower than that in other groups (P <0.01) The expression of HCCLM3 was significantly lower than that of other groups. There was no significant difference in the proliferation of HCCLM3 cells after transfection. The results of Matrigel invasion assay showed that the number of HCCLM3 cells passing through the artificial basement membrane decreased by 80% after transfection of S3, which was significantly lower than that of other groups (P <0.01). Conclusion The CCR1 expression of HCCLM3 in human hepatocellular carcinoma cells can be effectively inhibited by plasmid-introduced exogenous miRNAs, which in turn reduces the invasiveness of HCCLM3 cells but has no significant effect on cell proliferation.