The Role And Mechanisms Of MiR-154 Upregulating Wnt/?-catenin Pathway In Mouse Lung Fibroblast L929 Phenotype Transition

Research Objectives:1.To explore the expression of miR-154 in mouse fibroblast cell line L929 after induced by TGF-?1.2.To investigate the effect of miR-154 gene overexpressing or silencing on the biological characteristics of mouse fibroblast cell line L929.3.To initially explore the molecular mechanisms for the fibrotic roleof miR-154,which through increasing Wnt/?-catenin signal pathway in pulmonary fibrosis in vitro.Research method:1.Mouse fibroblast cell line L929 was used to establish an in-vitromodel of fibroblast-myofibroblst transition induced by TGF-?1.2.Divided cells into 5 groups:blank group,sh-NC group,MCS-SV40-NC group,shmiR-154 group and MCS-SV-miR-154 group. The-se five groups were transfected corresponding lentiviral vectors to establish in-vitro overexpressing or silencing model.3.miR-154 expression in five groups were measured by q RT-PCR after transfected by lentiviral.4.The cell activity was determined by CCK8 assay.The cell migration ability was detected by Would healing assay and Transwell assay.Flow cytometry was used to monitor the changes of cell cycle distribution.5.Western blotting and q RT-PCR were performed to assess the expression of?-SMA??-catenin?DKK2.Research results:1.TGF-?1(5ng/ml) was used to stimulate the mouse lung fibroblast L929 24 hours, then 12 hours to supplement once. The results of Western Blot showed that the expression level of ?-SMA was significantly increased(P<0.05). This showed that an in vitro model was constructed successfully.2.The display of cell transfection efficiency was observed by fluorescence microscopy. The lentiviral vectors successfully entered the L929. The expression level of miR-154 was detected by q RT-PCR, and the expression level of miR-154 was proved to be successfully constructed. The cell model of overexpressing miR-154 and inhibitoring miR-154 were successfully constructed.3.The expression of miR-154 in each group was detected by q RT-PCR method,then analysing the differences. The results show that the miR-154 expression level have no significant differences in the group of blank,sh-NC and MCS-SV40-NC.While the expression level of shmiR-154 significantly decreased and the miR-154 expression level of MCS-SV-miR-154 significantly increased.4.A series of cell function tests were performed on the 5 groups of cells:(1) The test result of CCK8:there have no signigicant differences among the blank, sh-NC and MCS-SV40-NC group. While the group of shmiR-154 has significantly decreased and cell proliferation of group MCS-SV-miR-154 shows significa-ntly increased.(2)Cell injury and healing experiment and Transwell:the decresased migration speed was seen in shmiR-154 whereas not in the presence of he blank,sh-NC and MCS-SV40-NC group. The group of MCS-SV-miR-154 was crawling with the fastest speed.(3) Then we examined the effects of miR-154 on cell-cycle progression in the mouse lung fibroblast L929 cells and observed a significant increasein S phase in MCS-SV-miR-154 group, while a significant decrease in the same phase in shmiR-154 group. There were no significant differences among the blank, sh-NC and MCS-SV40-NC group. It is suggested that the expression level of miR-154 did promote the transmission of the Wnt/?-catenin signal pathway, thus enhancing the biological characteristics of the mouse lung fibroblast L929 cell.5.Western blotting and q RT-PCR were performed to assess the expression of?-SMA ? ?-catenin ? DKK2: the gene expression of ?-catenin and ?-SMA in shmiR-154 group was down-regulated, and the expression was up-regulated in group MCS-SV-miR-154. The gene expression of DKK2 in shmiR-154 group was up-regulated,and the expression was down-regulated in group MCS-SV-miR-154.There were no significant differences among the blank, sh-NC and MCS-SV40-NC group.Research Conclusion:1. miR-154 expression was upregulated for stimulating by TGF-?1 in mouse lung fibroblasts cell line L929.2.miR-154 can activiate the expression of Wnt/?-catenin signaling pathway to promote the phenotypic transformation of mouse lung fibroblasts cell line L929.