miR-133a-3p重组腺相关病毒的构建及抑制LX-2细胞α-SMA基因表达

Construction of AAV8 that over -expresses miR-133a-3p and its inhibitory effect on α-SMA expression in LX-2 cells

目的构建miR-133a-3p重组腺相关病毒(recombinant adeno-associated virus, rAAV)过表达载体并转染肝星状细胞,鉴定其转染及干预作用。方法体外培养肝星状细胞株LX-2细胞,试验分为DMEM对照组,AAV8-Scrambled对照组,AAV8-miR-133a-3p组,应用qRT-PCR法检测miR-133a-3p转染表达量,检测胶原蛋白(COL1A1)、-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)和TGF-β/Smad信号通路基因的表达量。结果重组腺相关病毒成功构建并转染LX-2细胞,AAV8-miR-133a-3p转染LX-2细胞组的miR-133a-3p相对表达量为11 000±343.10,与DMEM对照组比较差异有统计学意义(t=32.07,P<0.01)。AAV8-miR-133a-3p转染组与DMEM组α-SMA mRNA的相对表达量分别为0.42±0.06 vs 1.00±0.09(t=5.72,P<0.05),COL1A1 mRNA的相对表达量为0.34±0.03 vs 1.00±0.04(t=12.66,P<0.01)。结论重组腺相关病毒成功转染LX-2细胞,且能抑制肝星状细胞纤维化标志物α-SMA的表达,为探讨miR-133a-3p在调控肝星状细胞所致纤维化中的作用奠定了理论基础。

Objectives To construct an AAV vector that over-expresses miR-133 a-3 p and to examine its inhibitory effect on α-SMA mRNA expression in the LX-2 hepatic stellate cell line. Methods LX-2 cells were cultured in vitro, divided into three groups, and transfected with three agents(DMEM control, AAV8-Scrambled group and AAV8-miR-133 a-3 p group). The relative expression of miR-133 a-3 p and the expression of α-SMA, COL1 A1, caspase 9, and TGF-β/Smad mRNA were detected using qRT-PCR. Results AAV8-miR-133 a-3 p was successfully constructed and transfected into LX-2 cells. miR-133 a-3 p was over-expressed in the AAV8-miR-133 a-3 p group. The relative level of expression of α-SMA mRNA in LX-2 cells transfected with AAV8-miR-133 a-3 p was 0.42±0.06. The level differed significantly from that in the DMEM control group(t=5.72, P<0.05). The relative level of expression of COL1 A1 mRNA was 0.34±0.03. The level differed significantly from that in the DMEM control group(t=12.66, P<0.01). There were no significant differences in expression of TGF-β/Smad mRNA(P>0.05). Conclusion AAV8-miR-133 a-3 p was successfully constructed and transfected into the LX-2 cell line. The results of inhibition of over-expression of miR-133 a-3 p indicated that AAV8-miR-133 a-3 p is a novel tool for further research on liver fibrosis regulated by HSC and miR-133 a-3 p.