利用RNA干扰技术抑制小鼠羟甲基戊二酸单酰辅酶A还原酶基因的表达

Inhibition of Hydroxymethylglutaryl CoA Reductase Gene Expression by RNA Interference in Mouse

目的为了探寻一种更有效的家族性高胆固醇血症(FH)基因治疗新途径,通过慢病毒介导RNA干扰的方法在H22细胞系及C57BL/6J纯系小鼠中抑制内源性羟甲基戊二酸单酰辅酶A还原酶(HMG-CoAR)基因表达进行研究。方法从HMG-CoAR基因序列中选择三段作为靶序列(T1、T2和T3),并构建重组慢病毒RNAi载体;经体外包装后,分别感染H22细胞系及注射小鼠。通过qRT-PCR检测HMG-CoAR mRNA丰度;利用WesternBlot、ELISA方法分析HMG-CoAR的表达水平。结果与空载体对照相比,三个实验组中稳定转染细胞的HMG-CoAR表达水平显著降低(P<0.05),同时感染5天后小鼠的肝脏HMG-CoAR mRNA水平、蛋白含量以及血浆中HMG-CoAR表达均显著降低(P<0.05),其中T3位点的抑制效率最高,是空质粒对照的1/3。另外,对三个载体的持续性抑制能力分析显示,在处理后的至少50天内,三个实验组小鼠中HMG-CoAR表达量均有效地被抑制,其中T1靶点的抑制效果最为稳定,而T3靶点的波动最大,但抑制效率一直最高。结论三个重组慢病毒RNAi载体均可显著抑制内源性HMG-CoAR表达,可为下一步试图通过下调HMG-CoAR表达的方式,降低FH患者体内胆固醇水平的FH基因治疗奠定基础。

Aim To,develop novel strategies to treat familial hypercholesterolemia （FH）, we explored knocking down the endogenous hydroxymethylg!utaryl CoA reductase （HMG-CoAR） expression in C57BL/6J mouse by lentivirus me- diated RNAi approach in this study. Methods Three lentiviral RNAi vectors targeting sites （T1, T2 and 13 ） in HMG-CoA reductase gene were subject to infect H22 cell line and tail vein injection to C57BL/6J mouse after viral packa- ging. HMG-CoAR mRNA abundance was measured by qRT-PCR; Western Blot and ELISA assay were used to detect the HMG-CoAR protein expression in infected mouse liver or plasma. Results The expression of HMG-CoAR in stably transfected H22 cells of three experimental groups was dramatically reduced in comparison to control （P 〈 0.05 ）. Significantly lowing HMG-CoAR was detected in mouse liver and plasma 5 days after infection （ P 〈 0.05 ）. Among the three target sites, T3 was proved to be the most effective, in which the HMG-CoAR level was only 1/3 of control group. Moreover, the result of time course analysis of the inhibition effects showed that the HMG-CoAR expression was supressed at least up to 50 days in all three groups. T1 site exhibited most stable inhibition effect, yet 33 site showed more fluctuating though it was more effective than others. Conclusion Three RNAi targeted vectors were demonstrated to be able to inhibit en- dogenous HMG-CoAR expression in cell line and mouse. The results pave the way for lowing serum lipid in FH treatment by knocking down HMG-CoAR level.