RNAi cyclin E对肝癌细胞生长的影响

The effect of RNAi cyclin E on growth of liver cancer cell

目的:探讨RNAi cyclin E对肝癌细胞生长的影响。方法:肝癌细胞系HepG2采用体外培养,选取siRNA合成双链发卡样DNA;将其重组入pGFP-V-RS得到重组子pGFP-V-RS-siCE951和pGFP-V-RSsiCE1122;两重组子分别进行DNA序列测定、同源性比较;将两重组子和pGFP-V-Con分别转入人肝癌HepG2细胞中,同时设空白对照组(不转染任何质粒,仅加转染试剂)。检测4组HepG2细胞cyclin E mRNA表达情况及细胞的增殖情况。结果:经过同源性检索和优选确定cyclin E基因的siRNA载体pGFP-V-RSsiCE951和pGFP-V-RS-siCE1122;干扰1组(转染pGFP-V-RS-siCE951)和干扰2组(转染pGFP-V-RSsiCE1122)细胞cyclin E mRNA的相对表达量显著低于空白对照组和无关siRNA对照组(P<0.05);干扰1组和干扰2组与空白对照组和无关siRNA对照组比较,在3、4、5 d细胞孔内的吸光度值显著减少,差异有统计学意义(P<0.05)。结论:siRNA载体pGFP-V-RS-siCE951和pGFP-V-RS-siCE1122转染人肝癌HepG2细胞能显著降低细胞cyclin E基因的表达,是理想的siRNA靶区段。抑制肝癌HepG2细胞cyclin E基因表达,可以降低肝癌HepG2细胞的生长速度。

Objective： To investigate the effects of RNAi cyclin E on growth of liver cancer cells. Methods： HepG2 cell line was cultured in vitro. The selected siRNA were formed into double- stranded hairpin DNA. Two recombinants of pGFP-V-RS-siCE951 and pGFP-V-RS-siCE1122 were obtained by insertion of hairpin DNA into pGFP-V-RS. DNA sequences were determined and ge- netic homology were compared in two recombinants. The two recombinants and pGFP-V-Con were transfected into HepG2 cells, the blank control group （added by transfection reagent without any transfection of plasmid） was set up. The expression of cyclin E mRNA and proliferation of HepG2 cells were tested in four groups. Results： Cyelin E gene siRNA vectors（pGFP-V-RS-siCE951 and pGFP-V-RS-siCE1122） were identified by homology search and optimization. The expression quantity of cyclin E mRNA in silencing group 1 （transfected by pGFP-V-RS-siCE951 ） and 2 （transfeeted by pGFP-V-RS-siCE1122） were significantly lower than unrelated siRNA and blank control groups （P 〈 0.05）. The absorbance in silencing group 1 and 2 significantly was decreased than two control groups in 3,4 and 5 days （P 〈 O. 05）. Conclusion ： The cyclin E siRNA vectors （pGFP-V-RS-siCE951 and pGFP-V-RS-siCE1122） could dramatically decreased the expression of cyclin E in HepG2 liver cancer cells by transfection, which were the ideal target of siRNA. The growth of HepG2 liver cancer cells could be controlled by inhibiting the gene expression of cyclin E.