沉默miR-216a对四氯化碳诱导的肝纤维化模型大鼠的保护作用及机制研究

目的探究沉默miR-216a对四氯化碳(CCl4)诱导的肝纤维化模型大鼠的作用及机制。方法从50只大鼠中随机选取10只作为正常组,其余40只采用腹腔注射含CCl4的橄榄油溶液方法构建肝纤维化模型大鼠。将造模成功的30只大鼠随机分为模型组、对照组和沉默组,每组各10只,对照组尾静脉注射含空载质粒的腺病毒,沉默组尾静脉注射含si-miR-216a质粒的腺病毒。采用酶联免疫吸附测定(ELISA)法检测各组透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(ⅣC)的表达水平。采用实时荧光定量聚合酶链反应(real-time qPCR)法检测miR-216a、第10号染色体缺失性磷酸酶及张力蛋白同源物基因(PTEN)mRNA和母亲DPP同源物7(Samd7)mRNA的相对表达量。采用蛋白质印迹法检测PTEN和Samd7的表达水平。结果与正常组比较,模型组HA、LN、PCⅢ、ⅣC和miR-216a表达水平均升高,而PTEN mRNA、Samd7 mRNA、PTEN和Samd7的表达水平均降低,差异均有统计学意义(P均<0.05)。与模型组和对照组比较,沉默组HA、LN、PCⅢ、ⅣC和miR-216a表达水平均降低,而PTEN mRNA、Samd7 mRNA、PTEN和Samd7表达水平均升高,差异均有统计学意义(P均<0.05)。结论沉默miR-216a可降低肝纤维化大鼠HA、LN、PCⅢ和ⅣC的表达水平,改善CCl4诱导的肝纤维化损伤,其机制可能与调控PTEN/Smad7信号通路有关。

Protective effect and mechanism of miR-216a silencing on carbon tetrachloride-induced liver fibrosis in rats

Objective This paper attempts to explore the effect and mechanism of microRNA(miRNA)216a silencing on carbon tetrachloride-induced liver fibrosis in rats.Methods Ten out of the fifty rats were randomly assigned to the normal group,and the remaining 40 rats were intraperitoneally injected with olive oil solution containing CCl4 to establish the liver fibrosis model rats.Thirty rats were randomly assigned to the model group,the control group,and the silent group,with 10 rats in each group.Adenovirus containing empty plasmid was injected into the control group through tail vein,while adenovirus containing plasmid si-miR-216a was injected into the silent group through tail vein.The levels of hyaluronic acid(HA),laminin(LN),typeⅢprocollagen(PCⅢ),and typeⅣcollagen(ⅣC)were detected by enzyme-linked immunosorbent assay.The expression of miR-216a,phosphatase,and tensin homolog deleted on chromosome 10(PTEN)mRNA and mothers against decapentaplegic homolog 7(Samd7)mRNA were detected by utilizing the real-time fluorescence quantitative PCR method.The expression of PTEN and Samd7 were detected by western blot.Results Compared with the normal group,the levels of HA,LN,PCⅢ,ⅣC,and miR-216a in the model group increased,while the levels of PTEN mRNA,Samd7 mRNA,PTEN,and Samd7 decreased,with statistically significant differences(P<0.05).Compared with the model group and the control group,the levels of HA,LN,PCⅢ,ⅣC,and miR-216a in the silent group decreased,while the levels of PTEN mRNA,Samd7 mRNA,PTEN,and Samd7 increased,with statistically significant differences(P<0.05).Conclusions miR-216a silencing can reduce the levels of HA,LN,PCⅢ,andⅣC in liver fibrosis rats and improve the liver fibrosis injury induced by carbon tetrachloride.The mechanism may be related to the regulation of PTEN/Smad7.