Cyclin D1-siRNA真核质粒的构建及对HepG2肝癌细胞增殖的影响

Construction of Cyclin D1 siRNA Vector and Effect on Proliferation of Human HepG2 Cancer Cells

目的 利用PGE-1建立针对抑制cyclinD1功能的siRNA 真核表达载体以及对肝癌HepG2细胞增殖和周期的影响.方法 针对cyclinD1 mRNA序列设计合成4对寡核苷酸,退火后连接到PGE-1质粒中,PCR及测序鉴定.用脂质体将重组质粒转染到HepG2中,RT-PCR cyclinD1 mRNA的表达,G418筛选后用RT-PCR和Westernblot技术分别检测cyclinD1 mRNA和蛋白质水平;流式细胞仪测定细胞周期的变化;MTT法测定细胞的生长.结果 在设计的4条靶序列中有一序列重组成质粒后,可明显抑制cyclinD1的表达;目的 序列表达载体转染HepG2细胞后,可以明显减少G2-M期细胞的比例,当HepG2细胞稳定转染针对cyclinD1的干扰质粒后,mRNA和蛋白质的表达也明显降低;HepG2细胞cyclinD1表达降低后,其生长也受到明显抑制.结论 成功构建针对siRNA-cyclinD1真核质粒,在体外可成功抑制人肝癌HepG2细胞的增殖.

Objective To explore the effect of cyclinDl gene blocking by siRNA on proliferation and cell cycle of hepG2 liver caicenoma cells. Methods Four pairs of DNA templates coding siRNA ,synthesized against cyclinDl and cloned into the vector PGE-l,were identified by restriction ndonuclease digestion analysis,PCR and DNA sequencing cells were then transfected with these four PGE-1-siRNAs and the negative control. After G418 selection,RT-PCR and Western blot were used to detect the expression of eyclinDl gene. Cell growth curve were drived by MTT assays. Results Restriction endonuclease digestion analysis and DNA sequencing results all showed that the target segments were cloned PGE-I vector respectively after the postive-siRNA was chosen to transfected into t tepG2 cells, the expression of cycllnDlmRNA and protein was marketly decreased. The results showed that after interfering,the ItepG2 cells cell growth were signifencantly inhibited. Conclusion The cyelinDl-speciflc siRNA mediated Dy PGE-1 could effectively knockdown tbe expression of gene and inhibits the proliferation potential ability of HepG2 cells.