siRNA沉默p62基因对人肝癌细胞Hep3B的生物学影响

Biological Effects of p62 siRNA on Hepatoma Cell Hep3B

目的:探讨siRNA沉默人肝癌细胞Hep3B中p62基因对人肝癌细胞的生物学影响。方法:人肝癌细胞Hep3B为靶细胞,siRNA通过Lipofectamine 2000转染Hep3B。首先从3条siRNA序列中筛选出1条沉默p62的最佳序列,然后将细胞分为空白对照组、siRNA组、Ncontro(lNC)组及脂质体(Lip)组。分别用RT-PCR和Western blot法检测各组细胞中p62、XPD、p53以及cyclinD1的mRNA和蛋白质的表达量,并用MTT法检测各组细胞的增殖能力。结果:siRNA组中p62、XPD、p53的mRNA表达较其他3组显著下调(P<0.01),而siRNA组中cyclinD1的mRNA表达较其他3组明显上调(P<0.01)。Westernblot检测结果显示各组细胞中p62、XPD、p53及cyclinD1的蛋白表达变化趋势与其mRNA变化趋势相一致。MTT检测显示siRNA沉默p62后,细胞增殖能力增强。结论:p62基因可能通过影响XPD、p53及cyclinD1从而抑制癌细胞生长,并且可能通过DNA损伤检控点来调控细胞增殖。

Objective： To investigate the biological effects of p62 siRNA on hepatoma cell Hep3B. Methods： Three siRNA sequences, specifically targeting p62 （named as Si-h-GTF2H1_001, Si-h-GTF2H1_002 and Si-h-GTF2H1_003）, were selected. The one of silent p62 was Si-h-GTF2H1_002. The siRNAs were transfected into hepatoma cell Hep3B with Lipofectamine 2000. There were four groups in this study including blank control group, siRNA group, Ncontrol （NC）group and Lipofectamine （Lip） group. The expressions of p62, XPD, p53 and cyclinD1 were detected by RT-PCR and Western blot. The cell cycle was examined by MTT. Results： Compared with blank control group, NC group and Lip group, the expressions of p62, XPD and p53 mRNA decreased significantly in siRNA group （P 0.01）; but the expressions of cyclinD1 mRNA was enhanced obviously in siRNA group（P 0.01）. The trends of p62, XPD, p53 and cyclinD1 protein were consistent with trend of mRNA detected by Western blot. MTT results showed that cell proliferation increased in siRNA group. Conclusion： p62 gene may inhibit cancer cells by interacting with XPD, p53 and cyclinD1, and possibly regulate cell proliferation through DNA damage checkpoint.