天津医药

• 论著 •    

siRNA沉默p62基因对人肝癌细胞Hep3B的生物学影响

罗小洋1,邬柏林2,胡桂梅3,罗忠金4   

  1. 1. 南昌大学第二附属医院消化科
    2. 南昌大学第二附属医院
    3. 宁波大学医学院附属医院
    4. 厦门市第二人民医院
  • 收稿日期:2009-12-01 修回日期:2010-02-05 出版日期:2010-06-15 发布日期:2010-06-15
  • 通讯作者: 罗小洋

Biological effects on hepatoma cell Hep3B of siRNA on expression p62

  • Received:2009-12-01 Revised:2010-02-05 Published:2010-06-15 Online:2010-06-15
  • Contact: 邬柏林 胡桂梅 罗忠金

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摘要： 【摘要】 目的：应用siRNA沉默人肝癌细胞Hep3B中p62基因的表达，探讨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最佳的序列。然后将细胞分为空白对照组、siRNA组、Ncontrol（NC）组、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的mRNA表达较空白对照组、NC组、Lip组显著性下调（P ＜0.01），siRNA组中xpd、p53的mRNA表达较其他3组也有明显的下调；而siRNA组中cyclinD1的mRNA表达较其他3组明显的上调。Western blot 检测结果显示各组细胞中p62、xpd、p53以及cyclinD1的蛋白质表达变化趋势与其mRNA 变化趋势相一致。MTT检测显示siRNA沉默p62后，细胞增殖力增强。结论：p62基因可能通过影响xpd、p53以及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。

关键词: 肝细胞癌, RNA干扰, 基因, p62, 转染, 细胞周期

Abstract: 【Abstract】 Abstract Objective:To investigate the biological effects on hepatoma cell Hep3B of p62 by use of p62 siRNA. Methods: First,Three siRNA sequences specifically targeting p62 (named as Si-h-GTF2H1_001, Si-h-GTF2H1_002 and Si-h-GTF2H1_003) were selected,the best one which silent p62 was Si-h-GTF2H1_002.The siRNAs were transfected into hepatoma cell Hep3B with Lipofectamine 2000.Then,the exprement were classified four groups: 1,blank control group；2,siRNA group；3,Ncontrol（NC）group；4,Lipofectamine（Lip）group.The expression of p62,xpd,p53,cyclinD1 were detected by RT-PCR and Western blot.The cell cycle were examined by MTT.Results : Contrast with blank control group , NC group, Lip group ,the expression of p62 mRNA was decreased significantly in siRNA group(p＜0.01）;the expression of xpd mRNA and p53 mRNA were decreased obviously;but the expression of cyclinD1 mRNA was enhanced obviously.The trends of protein was consistent with mRNA.MTT results showed that cell proliferation increased in siRNA group. Conclusion: p62 gene may initeract with xpd,p53 and cyclinD1 to inhibit cancer cell,and possibly regulate cell proliferation through DNA damage checkpoint.

Key words: hepatoma cell, small interference RNAs, gene, p62, transfection, cell cycle