Study on inhibiting expression of IGF1R in hepatocellular carcinoma cells by constructing shRNA eukaryotic expression vectors
- VernacularTitle:构建RNA干扰真核表达载体抑制肝癌细胞IGF1R表达的研究
- Author:
Chenfang ZHU
;
Qiang ZHONG
;
Yan GU
- Publication Type:Journal Article
- Keywords:
insulin like growth factor-1 receptor;
RNA interference;
eukaryotic expression vector;
hepatocellular carcinoma;
FAK
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2009;29(10):1163-1168
- CountryChina
- Language:Chinese
-
Abstract:
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-97 H 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 3, 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. Results The 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.
