微小RNA-29a通过沉默信息调节因子2相关酶类1基因调控肝细胞脂肪变性的机制

目的探讨人肝细胞株L02脂肪变性时微小RNA-29a(miR-29a)的表达变化及其靶向沉默信息调节因子2相关酶类1(silent mating type information regulation 2 homolog-1,Sirt1)调节脂肪肝细胞脂肪沉积的机制。方法采用油酸和棕榈酸混合物诱导建立非酒精性脂肪肝细胞模型,验证模型成功后,PCR检测miR-29a和Sirt1的表达变化;生物学预测miR-29a的靶基因;分别转染miR-29a模拟物和抑制剂,过表达miR-29a和抑制miR-29a后再建立人脂肪肝细胞模型。油红O染色观察细胞中脂质蓄积情况并测定甘油三酯含量,荧光定量PCR和免疫印迹法检测Sirt1基因和蛋白表达变化。结果脂肪肝细胞模型组miR-29a相对表达量和甘油三酯含量显著高于对照组(P<0.01),Sirt1相对表达量显著低于对照组(P<0.01);生物学预测Sirt1是miR-29a的靶基因,过表达miR-29a后,细胞内脂滴明显增多,脂肪沉积加重,甘油三酯含量显著增加(P<0.05),细胞中miR-29a的表达显著上调(P<0.01),而Sirt1 mRNA表达显著下调(P<0.05),Sirt1蛋白表达呈下降趋势;与之相反,抑制miR-29a后,细胞中脂滴相对减少,脂肪沉积减轻,甘油三酯含量显著下降(P<0.05),细胞中miR-29a的表达被有效抑制(P<0.01),而Sirt1 mRNA的表达显著上调(P<0.05),Sirt1蛋白表达较对照组呈上升趋势。结论 miR-29a在非酒精性脂肪肝细胞中表达显著上调,miR-29a通过表达上调负调控Sirt1表达从而促进脂肪肝细胞中脂肪沉积。

Mechanism of microRNA-29a regulating steatosis in the human hepatocyte via targeting silent mating type information regulation 2 homolog-1

OBJECTIVE To investigate the expression of microRNA-29 a(miR-29 a) in the human steatotic hepatocyte model and the mechanism of targeting silent mating type information regulation 2 homolog-1(Sirt1)to regulate fat deposition of steatotic hepatocyte. METHODS The nonalcoholic fatty liver cell model was induced by a mixture of oleic acid and palmitic acid. After successful validation model, the expression of miR-29 a and Sirt1 was measured by PCR. The target genes of miR-29 a was predicted in biological system. MiR-29 a mimic and miR-29 a inhibitor were transfected into hepatocytes, and then established the human steatotic hepatocyte model, the result of oil red O staining and triglyceride(TG)lipid content were observed, the expression of Sirt1 mRNA and protein were detected by qRT-PCR and Western blotting, respectively. RESULTS The steatosis hepatocyte model was successfully established. Compared with control group, the relative expression of miR-29 a and triglyceride increased significantly(P<0.01), while the relative expression of Sirt1 decreased significantly(P<0.01) in the model group. Sirt1 was a target gene of miR-29 a. After transfection, the lipid droplet and the deposition of fat increased obviously in miR-29 a mimic group than those in the control group. TG content in miR-29 a mimic group increased significantly(P<0.05), the expression of miR-29 a increased significantly(P<0.01), while the expression of Sirt1 mRNA decreased significantly(P<0.05), and the expression of Sirt1 protein showed a downtrend. On the contrary, after the inhibition of miR-29 a expression, the lipid droplets in miR-29 a inhibitor group were relatively reduced, the fat deposition was alleviated. The TG content was significantly decreased(P<0.05), the expression of miR-29 a in the cells was effectively inhibited(P<0.01), while the expression of Sirt1 mRNA was significantly increased(P<0.05), and Sirt1 protein was on an upward compared with the control group. CONCLUSION The expression of miR-29 a is significantly increased in the nonalcoholic fatty liver cell model. Upregulation of miR-29 a negatively regulates the expression of Sirt1, thus promoting fat deposition of steatotic hepatocyte.