FK-506 Down-Regulates MiR-429 To Inhibit Human Fibroblast Proliferation And Prevents Scar Adhesion

Objective:investigating the role of tacrolimus in inhibiting fibroblast proliferation by down-regulating miR-429 and its potential mechanism.Method:In order to determine the expression of miR-429 in postoperative scar tissue,we performed qRT-PCR detection of RNA extracted from human postoperative scar tissue.Human fibroblasts were cultured in vitro and treated with a solution of 0 μM and 50 μM in FK-506 for 24 h.Detection of the effect of tacrolimus on miR-429 in human fibroblasts by qRT-PCR.Predicting potential target gene of miR-429 PTEN via online miRNA targeting prediction database,then use Dual luciferase assay to determine the relationship of them.Fibroblasts treated with FK-506 were divided into concentration group(0,5,10,25,50,100 μM)and time group(0h,12 h,24h,36 h,48h,60h).CCK-8 assay for the effect of FK-506 on the proliferation of human fibroblasts cultured in vitro.Fibroblasts were treated differently and then grouped(control group,FK-506 group,miR-429 group,FK506 + miR-429 group,antimiR-429,FK506+antimiR-429).Edu proliferation assay was used to verify the effect of miR-429 on fibroblast proliferation.Finally,PTEN inhibitors were used to inhibit the expression of PTEN in fibroblasts.We use assay for cell proliferation,and Western-blot for detection of PTEN-related proteins and downstream proliferation-related protein expression in human fibroblasts.To determine the role of PTEN in proliferation.Result:qRT-PCR results showed that the miR-429 is significantly increased in normal human scar tissue after surgery and the level of miR-429 in human fibroblasts treated with 50μM FK-506 was lower than the control.Indicating that FK-506 can down-regulate the expression of miR-429 in human fibroblasts.The results of the co-transfection assay of the dual luciferase reporter gene showed that hsa-mir-429 inhibited the expression of the gene containing PTEN-3’UTR in 293 T cells,while no expression inhibition of the gene containing PTEN-Mut-3’UTR.It indicates that miR-429 can target the inhibition of PTEN gene expression in human fibroblasts.According to the results of CCK-8,the proliferation activity of the cells decreased with the increase of the concentration of FK-506,and the proliferation activity of the cells decreased with the prolongation of the action time of FK-506.In the results of Edu test,FK-506 can inhibit fibroblast proliferation.FK-506 and down-regulation of miR-429 can significantly reduce the number of fibroblast proliferation compared to the control group.When transfected miR-429 mimic into fibroblasts,the decrease of cell proliferation caused by FK-506 is partially reversed.The inhibitory effect of proliferation compared to the control group after partial use of PTEN inhibitor was partially attenuated.This indicates that down-regulation of miR-429 expression by FK-506 can inhibit the proliferation of fibroblasts.It shows that in Western-blot.After the cells were transfected with miR-429 mime,the results showed that the down-regulation of FK-506 and miR-429 could significantly increase the expression of PTEN compared with the control group,and the expression of Cyclin D1 was significantly reduced.Part of the miR-429 mimetic is reversed due to transfer into the cell.After using the PTEN inhibitor,the expression of CyclinD1 was increased compared with the control group,indicating that FK-506 can regulate the expression of PTEN and the downstream proliferation-related protein Cyclin D1 by down-regulating miR-429.Conclusion: FK-506 can prevent scar adhesion by down-regulating miR-429 inhibiting proliferation of human fibroblasts,and Its potential mechanism may be achieved by targeting the PTEN gene.