载脂蛋白C-Ⅲ基因沉默对HepG2细胞脂代谢的影响

Effects of the apolipoprotein C-Ⅲ gene silencing on lipid metabolism in HepG2 cells

目的:研究RNAi技术沉默载脂蛋白C-Ⅲ基因(ApoC3)表达后对HepG2细胞内外脂质含量及其他脂代谢相关基因表达的影响。方法:合成3对针对ApoC3mRNA不同位点序列的小干扰RNA(siRNA),并分组转染至人肝细胞株HepG2,实时荧光定量PCR和酶联免疫吸附法检测ApoC3mRNA及蛋白质的表达水平,筛选出沉默效果最佳的一对siRNA。将最有效的siRNA转染HepG2细胞,48h后测定细胞内外脂质含量以及脂代谢相关基因的mRNA表达水平。结果:25nmol/L siRNA和3μl/孔(24孔板)转染试剂的转染效率最高。ApoC3-siRNA-3对ApoC3基因的沉默效果最好,对mRNA的抑制率为94.6%,上清液中检测不到ApoC-Ⅲ。ApoC3-siRNA-3沉默ApoC3基因后细胞内三酰甘油(TG)和胆固醇含量显著高于对照组,细胞外TG含量显著低于对照组。与对照组比较,ApoC3-siRNA-3沉默组载脂蛋白B100基因mRNA表达水平显著增强,肝脂酶基因mRNA表达水平显著降低。结论:ApoC3基因沉默可显著升高HepG2细胞内TG含量及显著降低细胞外TG含量,其可能机制是通过减少极低密度脂蛋白(VLDL)颗粒的分泌以及增加VLDL残粒的摄取。

Objective：To investigate the effects of apolipoprotein C-Ⅲ gene （ApoCd） silencing by RNA interfering on the lipid contents in HepG2 cells or in the culture medium, and the expression patterns of the genes related to lipid metabolism. Method：Three pairs of small interfering RNA （siRNA） targeting different sequences of ApoC3 mRNA were synthesized and transfected into human hepatoma cell line HepG2 to screen the most effective siRNA. Real-time fluorescence quantitative RCR and enzyme-linked immunosorbent assay detection were employed to detect the expression of ApoC3 mRNA levels and apolipoprotein C-Ⅲ levels, respectively. The lipid contents in HepG2 cells or in culture medium and the expression patterns of the genes related to lipid metabolism were deter- mined at 48 hours after transfection with the most effective siRNA. Result：The combination of 25 nmol/L siRNA and 3 μl transfection reagent per well （24-well plate） got the highest transfection efficiency. By screening, ApoCd- siRNA-3 was found to be the most effective siRNA to silenee ApoC3 gene with the inhibition rate of 94.6 %for mRNA, and the non-detectable level for apolipoprotein C-Ⅲ in culture medium. The contents of triglycerides （TG） and cholesterol （Ch） were significantly higher in the cells treated with ApoCd-siRNA-3 than those in control cells. The content of TO was significantly lower in the culture medium of the cells treated with ApoCd-siRNA-3 than that in control. Regarding the gene expression patterns, the cells treated with ApoCd-siRNA-3 had signifi- cantly higher mRNA level of apolipoprotein B100 gene and significantly lower mRNA level of hepatic lipase gene eompared with that of the control cells. Conclusion：ApoC3 gene silencing can significantly increase the content of TG in HepG2 cells and significantly decrease the content of TG in culture medium. The possible mechanisms may be the reduction of the secretion of very low density lipoprotein （VLDL） particles and the elevation of the uptake of VLDL remnants.