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目的构建针对人RhoA基因的短发夹状双链RNA(shRNA)真核表达载体,观察其对人肝癌细胞株HepG2 RhoA表达的特异性抑制效应。方法设计合成针对RhoA mRNA编码序列两个不同靶点的核苷酸片断,并定向克隆到真核表达载体Pgenesil-2的U6启动子下,构建重组质粒phsRNA-RhoA1和pshRNA-RhoA2,不针对任何基因组序列的重组质粒pshRNA-HK作为阴性对照。脂质体法转染到HepG2细胞后,应用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹法(Western-blot)分别检测RhoA基因表达的抑制效应。结果酶切鉴定和测序结果证实重组质粒均构建成功。转染HepG2后,pshRNA-RhoA2组RhoA基因mRNA和蛋白的表达水平均显著降低,与pshRNA-HK组和空白细胞组相比,差异有统计学意义(r=-19.28,t=-7.08,P<0.05)。而pshRNA-RhoA1组抑制效应不明显,差异无统计学意义(r=-0.16,t=-0.30,P>0.05)。结论构建的重组质粒pshRNA-RhoA2能特异有效地抑制RhoA基因在肝癌细胞HepG2中的表达,为进一步研究RhoA基因在肝癌中的作用提供了有效的分子工具。
Objective To construct a short hairpin double stranded RNA (shRNA) eukaryotic expression vector targeting human RhoA gene and observe its specific inhibitory effect on human hepatocellular carcinoma cell line HepG2 RhoA. Methods Nucleotide fragments targeting two different targets of RhoA mRNA coding sequence were designed and synthesized and cloned into the U6 promoter of eukaryotic expression vector Pgenesil-2 to construct recombinant plasmids phsRNA-RhoA1 and pshRNA-RhoA2. The recombinant plasmid pshRNA-HK of the genomic sequence was used as a negative control. After transfected into HepG2 cells by Lipofectamine 2000, the inhibitory effect of RhoA gene expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot. Results Enzyme digestion and sequencing confirmed that the recombinant plasmids were constructed successfully. Compared with pshRNA-HK group and blank group, the expression level of RhoA mRNA and protein in pshRNA-RhoA2 group was significantly decreased after transfection with HepG2 (r = -19.28, t = -7.08, P <0.05). However, the inhibitory effect of pshRNA-RhoA1 group was not obvious, the difference was not statistically significant (r = -0.16, t = -0.30, P> 0.05). Conclusion The constructed recombinant plasmid pshRNA-RhoA2 can specifically and effectively inhibit the expression of RhoA gene in HepG2 cells, which provides an effective molecular tool for further studying the role of RhoA gene in hepatocellular carcinoma.