lncRNA锌指蛋白反义链1对四氯化碳诱导肝纤维化小鼠的作用

Study on the Effect of LncRNA ZFAS1 on Carbon Tetrachloride Induced Liver Fibrosis in Rats

目的 探讨长链非编码RNA (lncRNA)锌指蛋白反义链1(ZFAS1)在肝纤维化(LF)中的作用。方法 四氯化碳(CCl4)诱导小鼠LF模型。苏木精-伊红染色(H-E染色)和天狼猩红染色观察肝脏组织学改变,ELISA检测肝组织羟脯氨酸(HYP)含量,免疫组织化学(IHC)检测肝组织α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原α1(Col1α1)的表达,荧光定量PCR检测肝组织ZFAS1的表达水平,通过siRNA技术在NCTC1469细胞中沉默ZFAS1表达并模拟小鼠肝细胞的纤维化模型,免疫印迹法(Western-blot)检测细胞Col1α1、转化生长因子-β1(TGF-β1)、基质金属蛋白酶-2(MMP-2)的表达变化。结果 与对照组比较,CCl4诱导模型组小鼠肝组织内炎性细胞浸润明显,胶原纤维沉积明显增多,肝星状细胞(HSCs)活化增多(P<0.05),肝组织胶原分泌及其代谢产物HYP表达上调,肝组织内lncRNA ZFAS1表达明显升高(P<0.05)。与AML12细胞比较,NCTC1469细胞高表达内源性lncRNA ZFAS1(P<0.05);在NCTC1469细胞转染3种siZFAS1片段均可有效抑制ZFAS1表达(P<0.05),以siRNA-539下降最明显。采用TGF-β1处理NCTC1469细胞构建LF模型,Western-blot结果显示,与对照组比较,模型组的Col1α1、TGF-β1和MMP-2表达明显升高(P<0.05);与模型组比较,ZFAS1干扰+模型组的Col1α1、TGF-β1和MMP-2表达明显下降(P<0.05)。结论 干扰lncRNA ZFAS1对LF可能具有治疗作用。

Objective To investigate the role of zinc finger antisense 1(ZFAS1)in liver fibrosis(LF).Methods The LF model was established by CCl 4.Hematoxylin-eosin staining(H-E staining)and Sirius scarlet staining were used to observe the histological changes,and ELISA was performed to detect the hydroxyproline content in liver tissue.Immunohistochemistry(IHC)was performed to detect the expression ofα-smooth muscle actin(α-SMA)and typeⅠcollagenα1(Col1α1).Fluorescence quantitative PCR was performed to detect the expression level of ZFAS1.siRNA technology was used to silence ZFAS1 expression and the LF model was mimicked in NCTC1469 cells.The expression level of Col1αⅠ,transforming growth factor-β1(TGF-β1),matrix metalloproteinase-2(MMP-2)were detected by Western blot.Results Compared with the control group,the CCl 4-induced group showed significant increased inflammatory cell infiltration and collagen fiber deposition,increased activation of hepatic stellate cells(HSCs)(P<0.05),upregulated expression of collagen secretion and its metabolite hydroxyproline,and significantly higher expression of lncRNA ZFAS1 in liver tissue(P<0.05).Compared with AML12 cells,NCTC1469 cells highly expressed endogenous lncRNA ZFAS1(P<0.05).Transfection of all three siZFAS1 fragments in NCTC1469 cells effectively inhibited ZFAS1 expression(P<0.05),especially siRNA-539.The LF model was constructed by treating NCTC1469 cells with TGF-β1.Western-blot results showed that the expressions of Col1α1,TGF-β1 and MMP-2 were significantly higher in the model group compared with the control group(P<0.05),the expressions of Col1α1,TGF-β1,and MMP-2 were significantly decreased in the ZFAS1 interference+model group compared with the model group(P<0.05).Conclusion Inhibition of lncRNA ZFAS1 may have therapeutic effect on LF.