构建RNA干扰真核表达载体抑制肝癌细胞IGF1R表达的研究

Study on inhibiting expression of IGF1R in hepatocellular carcinoma cells by constructing shRNA eukaryotic expression vectors

目的构建胰岛素样生长因子-1受体(IGF1R)RNA干扰真核表达载体,探讨人肝癌细胞株MHCC-97H在IGF1R基因抑制前后黏附和侵袭能力的变化及相关信号分子的变化。方法以pGCsi-U6-Neo-GFP为空白质粒载体,以IGF1R为靶基因,按GeneBank IGF1R基因核苷酸序列和Tusch1设计原则,选择5对2条互补的带发夹结构的核苷酸序列,连接到pGCsi-U6-Neo-GFP空载体中。转化大肠杆菌Stable3,扩增后提取质粒,进行酶切鉴定和测序分析。转染模型细胞(293T),进行RT-PCR及Westernblotting检测,筛选出沉默效果最好的质粒载体。用脂质体转染MHCC-97H肝癌细胞,G418筛选并建立IGF1R基因沉默的MHCC-97H肝癌细胞株。检测MHCC-97H细胞株黏附和侵袭能力及FAK蛋白的表达变化。结果构建的IGF1R pGCsi-U6-Neo-GFP shRNA,经酶切和DNA测序证实与设计完全一致;RT-PCR及Western blotting检测发现IGF1R沉默效率达88%。MHCC-97H细胞IGF1R被沉默后黏附和侵袭能力下降,FAK蛋白表达下降。结论IGF1R pGCsi-U6-Neo-GFP shRNA能够降低MHCC-97H细胞黏附和侵袭能力并抑制FAK蛋白的表达。

Objective To construct short-hairpin RNA（shRNA） eukaryotic express vectors targeting of insulin like growth factor-1 receptor（IGF1R） gene,and to explore the changes of adhesion,invasion and FAK protein expression of MHCC-97H hepatocellular carcinoma cells with RNA interference. Methods The shRNA oligonucleotide fragments were designed and synthesized based on the sequence of IGF1R mRNA.Double strands were formed after annealing and inserted into pGCsi-U6-Neo-GFP vector.The recombinant was transformed into Stable ,then plasmids were extracted and identified by restriction enzyme and sequencing analysis.The most effective vectors were selected by RT-PCR and Western blotting after transfecting 293T cells.The best one was used to transform MHCC-97H cells which were selected with G418 to obtain positive colons.The changes of adhesion,invasion and FAK protein expression in MHCC-97H cells were studied. ResultsThe restriction enzyme analysis and sequencing analysis demonstrated that shRNA had been inserted into vectors,and their sequences were the same as the design.It was indicated by RT-PCR and Western blotting that the silencing efficacy of IGF1R was 88%.The ability of adhesion and invasion significantly decreased after IGF1R silencing in MHCC-97H cells,and so was the expression of FAK protein. Conclusion IGF1R pGCsi-U6-Neo-GFP shRNA can significantly decrease the abilities of adhesion and invasion in MHCC-97H cells,and inhibit the expression of FAK protein.