微小RNA-30a在肝纤维化中的表达及其影响被引量：5

MicroRNA-30a expression in liver fibrotic tissues and its influence on liver fibrosis

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摘要目的观察微小RNA（miRNA,miR）-30a在肝纤维化中的表达及其对肝纤维化的影响.方法通过C57BL/6小鼠胆管结扎（对照组10例,胆管结扎组15例）和10 ng/ml转化生长因子-β1（TGF-β1）处理肝星状细胞（LX-2和HSC-T6）分别构建体内和体外肝纤维化模型.通过转染miR-30a mimics和miR-30a agomir分别在肝星状细胞和小鼠体内过表达miR-30a.提取总RNA和全蛋白进行反转录-聚合酶链反应（RT-PCR）和Western blot实验检测miR-30a、α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（collagen Ⅰ）和基质金属蛋白酶抑制剂-1（TIMP-1）.通过苏木素-伊红（HE）染色、胶原纤维染色（Masson染色）检测肝组织纤维化程度.结果 RT-PCR检测10例对照组和15例胆管结扎组小鼠肝脏miR-30a相对表达水平（2.037 ±0.256,n=10;1.007 ±0.126,n=15;P＜0.01）.10 ng/ml TGF-β1处理肝星状细胞24h和48 hmiR-30a相对表达水平（LX-2：24 h,0.578±0.055,48 h,0.585±0.048;HSC-T6：24 h,0.698±0.055,48 h,0.610±0.033）,明显低于对照组（LX-2：1.013±0.080;HSC-T6：1.001±0.013,P＜0.05）,表明miR-30a在体内和体外诱导的肝纤维化模型中表达下降.通过miR-30a mimics转染肝星状细胞株诱导细胞内过表达miR-30a、α-SMA相对表达水平（LX-2：对照组1.024±0.106,miR-30a高表达组0.464±0.021,P＜0.05;HSC-T6：对照组1.031±0.095,miR-30a高表达组0.352±0.042,P＜0.05）.通过尾静脉注射miR-30a agomir在小鼠体内高表达miR-30a,胆管结扎预处理小鼠α-SMA相对表达水平（对照组2.618±0.116,miR-30a高表达组1.207±0.197,P＜0.05）.表明过表达miR-30a能够抑制体内和体外肝纤维化进程.结论 miR-30a在肝纤维化中表达下降,过表达miR-30a能抑制肝纤维化进程. Objective To observe the expression of microRNA （miRNA,miR）-30a in liver fibrotic tissues and its effect on liver fibrosis.Methods Hepatic stellate cells （HSCs：LX-2,HSC-T6） were treated with 10 ng/ml transforming growth factor （TGF）-β1,C57BL/6 mice were treated by bile duct ligation （BDL）（n =15）,and 10 mice were treated with sham as the control.MiR-30a mimics and agomir were transfected into HSCs and mice,respectively.The expression levels of miR-30a,α-smooth muscle actin （o-SMA）,collagen Ⅰ,the tissue inhibitor of metalloproteinase 1 （TIMP-1） in vivo and in vitro were measured by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting.We detected the degree of liver fibrosis by hematoxylin and eosin （HE） and Masson staining.Results RT-PCR showed that miR-30a expression level was significantly lower in mice liver fibrotic tissues （1.007 ± 0.126,n =15） than in the normal liver tissues （2.037 ± 0.256,n =10）（P 〈 0.01）.LX-2and HSC-T6 cells were treated with TGF-β1 （10 ng/ml） for 0,12,24 and 48 h,respectively.The miR-30a expression showed a time-dependent decrease by TGF-β1 in HSCs （LX-2：0 h,1.013 ± 0.080,24 h,0.578±0.055,48 h,0.585 ±0.048;HSC-T6：0 h,1.001 ±0.013,24 h,0.698 ± 0.055,48 h,0.610 ±0.033;P 〈0.05）.These results indicated that miR-30a was significantly down-regulated in activated HSCs induced by TGF-β1 and in mice liver fibrotic tissues induced by BDL.α-SMA mRNA expression was detected in HSCs （LX-2：miR-NC 1.024 ± 0.106,miR-30a0.464±0.021,P〈0.05;HSC-T6：miR-NC 1.031±0.095,miR-30a0.352±0.042,P〈0.05） and in mice liver tissues （BDL ± AC 2.618 ± 0.116,BDL ± miR-30a 1.207 ± 0.197,P 〈 0.05）.These results showed that over-expression of miR-30a could inhibit fibrosis process in vivo and in vitro.Conclusion MiR-30a is down-regulated in liver fibrosis and up-regulation of miR-30a prevents liver fibrogenesis.