| Objective To investigate the role of miR-497-5p in regulating Reck in TGF-β1 induced hepatic stellate cells activation.Methods 1.HSC-T6 cells were cultured in vitro and stimulated with 5ng/ml,10ng/ml,15ng/ml and 20ng/ml TGF-β1 for 24 h,48h and 72 h,respectively,and cell viability was detected by CCK8 method to select the best concentration and time to establish a Hepatic stellate cells activation model.2.HSC-T6 cells were transfected with miR-497-5p mimics,mimics NC,miR-497-5p inhibitor and inhibitor NC,and the transfection efficiency of miR-497-5p was verified by QPCR.3.The experiment was randomly divided into: normal control group,TGF-β1 induction group(model group),TGF-β1+miR-497-5p mimics transfection group,TGF-β1+mimics NC transfection group,TGF-β1+miR-497-5p inhibitor transfection group,TGF-β1+inhibitor NC transfection group.4.CCK8 method was used to detect the cell viability of each group,m RNA expression levels of miR-497-5p,α-SMA,Col-I,MMP2,MMP9 and Reck in each group were detected by QPCR,and protein expression levels of α-SMA,Col-I,MMP2,MMP9 and Reck in each group of cells by Western-Blot assay.Results 1.Different concentrations of TGF-β1 and different time induced the viability of HSC-T6 cells after activation.The results showed that the viability and proliferation of HSC-T6 cells were the highest after 24 h intervention of 15ng/ml TGF-β1.2.The transfection efficiency was verified by QPCR.The results showed that compared with the mimics NC group,the relative expression level of miR-497-5p in the miR-497-5p mimics group was significantly increased(P<0.001);Compared with inhibitor NC group,the relative expression of miR-497-5p decreased significantly in miR-497-5p inhibitor group(P<0.0001).3.CCK8 was used to detect the cell viability of the intervention group,and the results showed that the cell viability of the model group was increased compared with the normal control group(P<0.05);Compared with the model group,cell viability of miR-497-5p mimics group was increased(P<0.05),cell viability of miR-497-5p inhibitor group was decreased(P<0.01);Compared with miR-497-5p mimics group,cell viability was decreased in the mimics NC group(P<0.01),cell viability of miR-497-5p inhibitor group was decreased(P<0.01);Compared with miR-497-5p inhibitor group,cell viability was increased in inhibitor NC group(P<0.01).4.m RNA expression levels of miR-497-5p,α-SMA,Col-I,MMP2,MMP9 and Reck in the intervention groups.The results showed that compared with the normal control group,the expression level of miR-497-5p in the model group was increased(P<0.05),m RNA expression levels of α-SMA,Col-I,MMP2 and MMP9 were significantly increased(P<0.01),the m RNA expression of Reck was decreased.Compared with the model group,the expression of miR-497-5p in the miR-497-5p mimics NC group was significantly increased(P<0.05),m RNA expression levels of α-SMA,MMP2 and MMP9 were increased(P<0.01),m RNA expression of Col-I was increased(P<0.05),the m RNA expression of Reck was decreased(P<0.01);miR-497-5p inhibitor group miR-497-5p expression was decreased(P<0.05),α-SMA and MMP2 m RNA expression were decreased(P<0.05),the m RNA expression levels of Col-I and MMP9 were significantly decreased(P<0.01),the m RNA expression of Reck was significantly increased(P<0.01).Compared with miR-497-5p mimics group,miR-497-5p expression was decreased in mimics NC group and miR-497-5p inhibitor group(P<0.05),in the mimics NC group the m RNA expression levels of α-SMA,Col-I,MMP2 and MMP9 were decreased significantly(P<0.01),the m RNA expression of Reck was up-regulated;in the miR-497-5p inhibitor group α-SMA,Col-I,MMP2 and MMP9 m RNA expression levels were decreased significantly(P<0.01),the m RNA expression of Reck was up-regulated(P<0.01).Compared with miR-497-5p inhibitor group,miR-497-5p expression was increased in inhibitor NC group,but there was no statistical significance.The m RNA expression levels of α-SMA,Col-I,MMP2 and MMP9 was increasedin inhibitor NC group,but there was no statistical significance,the m RNA expression of Reck was increased(P<0.01).miR-497-5p was negatively correlated with the relative expression level of Reck m RNA(r=-0.58,P=0.012).5.The protein expression levels of α-SMA,Col-I,MMP2,MMP9 and Reck in each intervention group.The results showed that compared with the normal control group,α-SMA,Col-I,MMP2 and MMP9 protein expressions were increased in the model group(P<0.01).Compared with the model group,the protein expressions of α-SMA,Col-I,MMP2 and MMP9 in miR-497-5p mimics group were increased(P<0.05);miR-497-5p inhibitor group α-SMA protein expression was decreased(P<0.05),the protein expressions of Col-I,MMP2 and MMP9 were decreased(P<0.01),the Reck protein expression was increased(P<0.05).Compared with miR-497-5p mimics group,the protein expressions of α-SMA,Col-I,MMP2 and MMP9 were decreased in miR-497-5p inhibitor group(P<0.01),the protein expression of Reck was increased(P<0.01);Col-I protein expression was decreased in mimics NC group(P<0.05),the protein expressions of MMP2 and MMP9 were decreased(P<0.01).Compared with miR-497-5p inhibitor group,protein expressions of MMP2 and MMP9 were increased in inhibitor NC group(P<0.01),Reck protein expression was decreased.Conclusion miR-497-5p was negatively correlated with the expression of Reck gene,and the up-regulated expression of miR-497-5p increased the activity of HSC-T6 cells and the expression of α-SMA and Col-I,which could promote the development of liver fibrosis.Down-regulating the expression of miR-497-5p,decreased the activity of HSC-T6 cells,and decreased the expression of α-SMA and Col-I,which can inhibit the development of liver fibrosis.Inhibition of miR-497-5p can increase the expression of Reck and reduce the expression of downstream genes MMP2 and MMP9,which may play an anti-fibrosis role by inhibiting the activation of HST-T6 cells. |
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