| Objective To explore the expression levels of miR-188-5p in keloid(KD),hypertrophic scar(HS)and normal skin(NS)tissue and fibroblasts.To investigate the value of miR-188-5p in the diagnosis and treatment of keloid and hypertrophic scar.To validate miR-188-5p's effects on the proliferation,migration and invasion of keloid and hypertrophic scar fibroblasts.To research the regulatory relations in order to explore the effection on related signaling pathways.Method Fibroblasts were fostered from the tissues of KD,HS and normal skin which having been diagnosed,and then identified the cells with immunofluorescence.Quantitative real-time polymerase chain reaction(qRT-PCR)were applied to detect the relative expression of miR-188-5p in tissues and fibroblasts of 9 KD patients,9 HS patients and 9 healthy normal controls.It was used by transfecting cells with chemically synthesized oligonucleotides complementary of miR-188-5p to loss and gain of function.Furthermore,cell proliferation,migration and invasion were checked in keloid fibroblasts(KFs)and hypertrophic scar fibroblasts(HSFs)respectively by Cell Counting Kit-8(CCK-8)assay,EdU imaging assay,Wound healing assay and Transwell assay.The interrelated proteins of MMP-2,MMP-9,Akt and p-Akt were ascertained by Western blot assay.Result(1)The expression level of miR-188-5p was significantly down-regulated in KD tissues(P<0.05),but there was no difference expression in HS and NS tissues.Consistent with the results obtained for tissues,miR-188-5p was remarkably down-regulated in KFs(P<0.01),compared with HSFs and normal skin fibroblasts(NFs).(2)It was demonstrated that enhancement of miR-188-5p stimulated obvious inhibition of KFs and HSFs proliferation after 72 h transfection by CCK-8 assay(P<0.01).In contrast,the downregulation of miR-188-5p induced significant promotion of KFs and HSFs proliferation at 72 h after transfection(P<0.01).(3)In the mimic-KFs group,the incorporating rate was 17.8 percent,and was significantly lower comparing the control group(54.2% and 53.8%)(P<0.01).Contrarily in the HSFs transfected with miR-188-5p inhibitor,the cell banding rate was 61 percent which was obviously higher than the control group(34.8% and 32.8%)(P<0.01).(4)Enhancing expression of miR-188-5p in KFs was correlated with significantly slow wound healing after 48 h(P<0.01),whereas lessened the expression of miR-188-5p in HSFs leading to significantly faster wound closure(P<0.01).(5)Transwell assays with Matrigel to demonstrate that KFs miR-188-5p-mimic had a lower invasive activity than KFs control,scramble control cells(p<0.01).By contraries,HSFs miR-188-5p-inhibitor showed a higher invasive activity than control,negative control cells(p<0.01).(7)The protein levels of MMP-2,MMP-9,PI3 K and p-Akt in miR-188-5p-mimic transfected KFs were obviously repressed compared with the scramble control transfection(P<0.05).In opposite,after transfecting miR-188-5p inhibitor,the protein levels of MMP-2,MMP-9,PI3 K and p-Akt were higher than the control(P<0.05).Conclusion The expressions of miR-188-5p in KD tissues and fibroblasts were significantly down-regulated than that in HS and normal skin,but there was no difference in the relative expression of mi R-188-5p in HS and normal tissues and cells.This discovery comfirmed that miR-188-5p may play an important role on the diagnosis and treatment of KD.Aberrant expression of miR-188-5p suppressed KFs proliferation,migration and invasion via regulated the expressions of MMP-2/9 and the signaling of PI3K/Akt,meanwhile,it promoted HSFs to gain the ability of invasion.These findings conjointly demonstrate a tumor suppressive role of miR-188-5p in KD proliferation and invasion via PI3K/Akt/MMP-2/9 signaling pathway,testifying that miR-188-5p could act as a new potential prognostic marker and therapeutic target for KD. |
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