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
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摘要
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.
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.
引文
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[2] Kitano M,Bloomston PM.Hepatic stellate cells and micrornas in pathogenesis of liver fibrosis[J].J Clin Med,2016,5(3):E38.
[3] Feng Y,Niu LL,Wei W,et al.A feedback circuit between miR-133 and the ERK1/2 pathway involving an exquisite mechanism for regulating myoblast proliferation and differentiation[J].Cell Death Dis,2013,4(11):e934.
[4] Puche JE,Saiman Y,Friedman SL,et al.Hepatic stellate cells and liver fibrosis[J].Compr Physiol,2013,3(4):1473-92.
[5] Arriazu E,Ruiz de Galarreta M,Cubero FJ,et al.Extracellular matrix and liver disease[J].Antioxid Redox Signal.2014,21(7):1078-97.
[6] Yoshida K,Murata M,Yamaguchi T,et al.TGF-β/Smad signaling during hepatic fibro-carcinogenesis[J].Int J Oncol,2014,45(4):1363-71.
[7] Gordon KJ,Blobe GC.Role of transforming growth factor-beta superfamily signaling pathways in human disease[J].Biochim Biophys Acta,2008,1782 (4):197-228.
[8] 赵卫华,王燕红,丛敏.转化生长因子β对肝星状细胞活化的调控作用[J].临床肝胆病杂志,2016,32(11):2195-98.
[9] Sanjay S,Girish C.Role of miRNA and its potential as a novel diagnostic biomarker in drug-induced liver injury[J].Eur J Clin Pharmacol,2017,73(4):399-407.
[10] Duan LJ,Qi J,Kong XJ,et al.MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response:implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction[J].Cell Signal,2015,27(2):215-27.
[11] Wang L,Li X,Zhou Y,et al.Downregulation of miR-133 via MAPK/ERK signaling pathway involved in nicotine-induced cardiomyocyte apoptosis[J].Naunyn Schmiedebergs Arch Pharmacol,2014,387(2):197-206.
[12] Roderburg C,Luedde M,Vargas Cardenas D,et al.miR-133a mediates TGF-β-dependent derepression of collagen synthesis in hepatic stellate cells during liver fibrosis[J].J Hepatol,2013,58(4):736-42.
[13] Ohno K,Ito M,Kawakami Y,et al.Collagen Q is a key player for developing rational therapy for congenital myasthenia and for dissecting the mechanisms of anti-MuSK myasthenia gravis[J].J Mol Neurosci,2014,53(3):359-61.