siRNA 抑制 HM GA1基因表达对 LX-2细胞生物学功能的影响

目的：探讨高迁移率蛋白 A1（HMGA1）siRNA 对人肝星状细胞 HMGA1、α-SMA 、E-钙黏素（E-cadherin）基因的表达调控作用及增殖活性的影响，并探讨其可能的机制。方法将有效的人工合成的 HMGA1 siRNA 转染人肝星状细胞 LX-2（LX-2）后沉默 HMGA1基因的表达。通过实时荧光定量 PCR（RT-PCR）及蛋白免疫印迹法（Western blot）检测 LX-2细胞中HMGA1、α-SMA 和 E-cadherin 的 mRNA 及蛋白表达水平。采用四甲基偶氮唑盐（M TT ）法检测 LX-2细胞增殖水平。结果以HMGA1-siRNA 序列1组沉默效果最好。 TGF-β1刺激组与 TGF-β1＋ NC-siRNA 组之间细胞增殖水平、HMGA1、α-SMA 、E-cad-herin 的基因及蛋白表达水平差异无统计学意义（P ＞0．05），细胞增殖水平、HMGA1、α-SMA 表达均明显高于正常对照组（P ＜0．05），E-cadherin 表达显著低于正常对照组（P＜0．05）；TGF-β1＋ HMGA1 siRNA 组细胞增殖水平及 HMGA1、α-SMA 的 mR-NA 及蛋白表达水平较另外3组均显著下降（P＜0．05），E-cadherin 表达水平显著增高（P＜0．05）。结论靶向 HMGA1的 siRNA能够沉默 LX-2中 HMGA1的表达；抑制 TGF-β1诱导的 LX-2增殖水平，提示 HMGA1参与了 TGF-β1诱导的肝星状细胞活化。

Effects of siRNA inhibit HMGA1 gene expression on LX-2 cell biological functions

Objective To investigate the effects of siRNA mediated HMGA 1 silence on proliferation and the gene expression of HMGA1 ,α-SMA and E-cadherin in activated hepatic stellate cells and its mechanisms .Methods Synthetic HMGA1 siRNA was transfected into LX-2 cells to silence the HMGA1 gene .The expression level of HMGA1 ,α-SMA and E-cadherin was determined by RT-PCR and Western blot experiments .LX-2 cell proliferation was assessed by M TT assay .Results The best inhibited effect was HMGA1-siRNA-1 .Compared with control group ,the cell proliferation and the mRNA and protein expression of HMGA 1 ,α-SMA in TGF-β1 group and TGF-β1 + NC-siRNA group were significantly increased (P< 0 .05) ,without significant differences between the two groups (P> 0 .05) ,while the expression of E-cadherin in TGF-β1 group and TGF-β1 + NC-siRNA group were significantly decreased compared with control group (P< 0 .05) .Meanwhile ,the cells in TGF-β1 + HMGA1 siRNA group showed significantly decreased proliferation level ,down-regulated mRNA and protein expression of HMGA 1 ,α-SMA but up-regulated expression of E-cadherin compared with TGF-β1 group and TGF-β1 + NC-siRNA group(P< 0 .05) .Conclusion HMGA1 interference could signifi-cantly down-regulate the expression of HMGA1 in LX-2 cells cultured with TGF-β1 ,thus inhibiting the proliferation and activation of the cells .