Construction and Screening of shRNA Expression Plasmids Targeting at cortactin Gene
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摘要: 目的 构建靶向cortactin基因的shRNA重组质粒，并筛选出沉默cortactin基因效果最明显的shRNA重组质粒。方法 设计并合成3对针对cortactin基因不同位点的shRNA片段，构建相应的重组质粒（命名为pBSilence1.1-cortactin-shRNA1-3），并进行酶切鉴定和测序分析。通过脂质体转染方法将各重组质粒及阴性对照质粒分别转染至人肝癌HepG2细胞中，转染24 h后荧光显微镜下观察各组细胞的转染情况，并采用RT-PCR检测各组细胞mRNA的表达情况。结果 构建的重组质粒经酶切鉴定与测序分析证实完全符合设计要求, 转染成功的细胞在荧光显微镜下显示绿色荧光，3组重组质粒组HepG2细胞的cortactin基因mRNA表达水平明显低于阴性对照组和空白对照组（P<0.05）。与pBSilence1.1-cortactin-shRNA1和pBSilence1.1-cortactin-shRNA2两组相比，pBSilence1.1-cortactinshRNA3组的cortactin基因mRNA表达降低更加显著（P<0.05）。结论 成功构建并筛选的重组质粒能有效抑制HepG2细胞中cortactin基因mRNA表达，为下一步研究沉默cortactin基因对肝癌细胞生物学行为的影响提供了实验基础。Abstract: Objective To construct short hairpin RNA (shRNA) expression plasmids targeting at the cortactin gene, and to screen the plasmid with the most knockdown efficiency. Methods Three pairs of shRNAs targeting at the cortactin gene were designed and synthesized. The shRNA expression plasmids (named pBSilence1.1-cortactin-shRNA1-3) were constructed and identified using restriction enzyme analysis and sequence analysis. The plasmids (pBSilence1.1-cortactin-shRNA1-3 and negative control plasmid) were then transfected into HepG2 cells via liposome. The transfection results were observed after 24h, and the cortactin mRNA expression was measured by reverse transcriptase-polymerase chain reaction(RT-PCR). Results The expression plasmids were confirmed by restriction enzyme analysis and sequence analysis. After the plasmid vector with green fluorescent protein was transfected to the cells, it was seen as a green fluorescent wave. RTPCR results showed that the cortactin mRNA expression in HepG2 cells of the three recombinant plasmids groups were decreased significantly compared with the negative control and blank control group(P＜0.05). The pBSilence1.1-cortactin-shRNA3 had the strongest knockdown efficiency compared with pBSilence1.1- cortactin-shRNA1 and pBSilence1.1-cortactin-shRNA2 groups(P＜0.05). Conclusion The constructed and screened shRNA expression plasmid targeting at the cortactin gene could suppress the cortactin mRNA expression in HepG2 cells significantly, which provides an experimental basis to study the effect of cortactin gene silencing on the biological behavior of liver cancer cells by RNA interference.
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Key words:
- Cortactin /
- Short hairpin RNA(shRNA) /
- RNA interference /
- Liver cancer
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