| Objectives To explore the effects of Wnt5 a on the proliferation and migration of hepatic stellate cells(HSCs),and to determine the mi R-194-5p targeting Wnt5 a and the effect of mi R-194-5p on HSCs,so as to provide a new target for the diagnosis,prevention and treatment of hepatic fibrosis(HF).Methods 1 Western blot,immunofluorescence and real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)were used to detect the expression of α-SMA,COL1A1 and Wnt5 a in LX-2 cells treated with TGF-β1.2 CCK8,colony formation,wound healing,Transwell and senescence β-galactosidase assay were used to detect the proliferation,colony formation,migration and senescence of LX-2 cells transfected with si-Wnt5 a.3 mi RNAs expression profile chip analysis of differentially expressed mi RNAs in liver tissue of rats in sham-operated group and common bile duct ligation group with hepatic fibrosis.4 The mi RNAs target Wnt5 a was predicted by Target Scan,mi RWalk and ENCORI websites,and verified by double luciferase reporter gene experiment.5 q RTPCR was used to detect the expression of mi R-194-5p in LX-2 cells treated with TGF-β1.6 q RT-PCR and Western blot were used to detect the m RNA and protein expression of α-SMA,COL1A1 and Wnt5 a in LX-2 cells transfected with mi R-194-5p mimics or mi R-194-5p inhibitor.7 CCK8,colony formation,wound healing,Transwell and senescence β-galactosidase assay were used to determine the proliferation,colony formation,migration and senescence of LX-2 cells transfected with mi R-194-5p mimics or mi R-194-5p inhibitor.Results 1 The expression levels of α-SMA,COL1A1 and Wnt5 a m RNA and protein in LX-2 cells treated with TGF-β1(10 ng/m L),the fluorescence intensity of α-SMA and Wnt5 a,and the expression levels of α-SMA and Wnt5 a m RNA in NFs cells treated with TGF-β1(10 ng/m L)were significantly higher than those in their respective control groups,and the differences were all statistically significant(P<0.05).2 Compared with the control group,the expression levels of Wnt5 a m RNA and protein in LX-2 cells transfected with si-Wnt5a-1,si-Wnt5a-2 and si-Wnt5a-3 were significantly lower,and the differences were statistically significant(P<0.05).3 Compared with the control group,the m RNA and protein expression levels of α-SMA,COL1A1 and Wnt5 a in LX-2 cells in siWnt5a-2 and si-Wnt5a-3 groups were decreased,and the differences were statistically significant(P<0.05).4 Compared with the control group,the proliferation of LX-2 cells,colony formation and migration ability of LX-2 cells in si-Wnt5a-2 and si-Wnt5a-3 groups were decreased,and the senescence degree of LX-2 cells was increased,and the differences were statistically significant(P<0.05).5 mi RNAs microarray was used to analyze the differentially expressed mi RNAs in the liver tissues of rats in sham operation group and choledochal ligation hepatic fibrosis group,and the down-regulated mi RNAs in the follow-up study was screened.mi R-194-5p was predicted to target Wnt5 a by bioinformatics software: Target Scan,mi RWalk and ENCORI.The luciferase activity of LX-2 cells in each group was determined by double luciferase reporter gene assay.Compared with the control group,the luciferase activity of wild-type fluorescent reporter vector and mi R-194-5p mimics co-transfection group decreased significantly,the difference was statistically significant(P<0.05),but there were no significant change in mutant fluorescent reporter vector and mi R-194-5p mimics co-transfection group.6 The level of mi R-194-5p in LX-2 cells and NFs cells treated with TGF-β1(10ng/m L)were significantly lower than that in the control group,and the differences were statistically significant(P<0.05).7 After transfection of mi R-194-5p mimics and mi R-194-5p inhibitor,the expression of mi R-194-5p increased in the former and decreased in the latter,the differences were statistically significant(P<0.05).8 mi R-194-5p inhibited the m RNA and protein expression of α-SMA,COL1A1 and Wnt5 a in LX-2 cells,and the differences were statistically significant(P<0.05).9 mi R-194-5p inhibited the proliferation,colony formation and migration of LX-2 cells,and promoted the senescence of LX-2 cells,and the differences were statistically significant(P<0.05).Conclusions 1 Silencing Wnt5 a inhibited the activation of LX-2 cells and promoted the senescence of LX-2 cells.2 Wnt5 a was the direct target gene of mi R-194-5p.3 mi R-194-5p could inhibit the activation of LX-2 cells and promote the senescence of LX-2 cells.Figure 36;Table 4;Reference 143... |
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