MiR-29a-3p Targeting COL3A1 Regulates The Proliferation And Apoptosis Of Keloid Fibroblasts

BackgroundKeloid disease（KD）is the product of abnormal wound healing after skin injury.It often has symptoms such as pain or pruritus,affects beauty and leads to local dysfunction,which seriously affects the quality of life of patients.The formation mechanism of KD is still unclear.Current treatment methods have limited efficacy and are prone to relapse.Therefore,new treatment strategies need to be explored.Micro RNA（miRNA）is a kind of short non-coding RNA that regulates the expression of a variety of genes in vivo.With the deepening of KD research,many miRNAs have been proved to be the key regulators in the pathogenesis of KD.Collagen Ⅲ alpha-1（COL3A1）is a type of collagen gene,which is involved in the encoding of Collagen Ⅲ α-1 chain and has been proved to be elevated in KD.This study explored the role and preliminary mechanism of miR-29a-3p and COL3A1 in KD.Objective1.To observe the expression of miR-29a-3p in keloid;2.To prove the targeting effect of miR-29a-3p on COL3A1 in keloid fibroblasts;3.To observe the regulatory effect of miR-29a-3p on the proliferation and apoptosis of keloid fibroblasts.Methods1.A total of 28 clinically confirmed scar tissues from patients with keloid and 15 surgically removed normal skin tissues were collected.KD diagnosis was confirmed by HE staining and Quantitative reverse transcription-PCR（qRT-PCR）was performed to detect the expression levels of miR-29a-3p and COL3A1 in scar tissue and normal dermal tissue.2.Observe the effect of up-regulated miR-29a-3p expression on KFBs: transfer miR-29a-3p mimic or miR-NC into human keloid fibroblasts（KFBs）,detect the transfection effect by qRT-PCR,cell proliferation and apoptosis were detected by CCK-8and flow cytometry.3.Prove that miR-29a-3p targets COL3A1: the potential target gene COL3A1 of miR-29a-3p was predicted by Targetcan,KFBs cells were co-transfected with wild-type reporter gene vector（containing pmir GLO-Wt-COL3A1 3’UTR）or mutant reporter gene vector（containing pmir GLO-Mt-COL3A1 3’UTR or pmir GLO-M1t-COL3A1 3’UTR or pmir GLO-M2t-COL3A1 3’UTR）and miR-29a-3p mimic or miR-NC,respectively.The targeting relationship between miR-29a-3p and COL3A1 was verified by dual luciferase reporter assay and Western blot.4.The influence of overexpression of COL3A1 on the up-regulated miR-29a-3p KFBs cells effect was detected by rescue experiment: miR-29a-3p mimic or miR-29a-3p mimic + pc DNA3 1-COL3A1 or miR-NC were transfected into cells respectively.The expression of COL3A1 in each group was detected by Western blot.CCK-8 method and flow cytometry were used to observe the effects of overexpression of COL3A1 on the proliferation and apoptosis of KFBs cells.Results1.Compared with normal tissues,the expression level of miR-29a-3p decreased significantly（P < 0.01）,and the expression level of COL3A1 increased significantly（P <0.01）;2.The results of qRT-PCR and Western blot showed that the expression of miR-29a-3p increased significantly and the expression of COL3A1 protein decreased significantly（P < 0.01）.CCK-8 method and flow cytometry showed that the up-regulation of miR-29a-3p expression inhibited the cell proliferation of KFBs and promoted cell apoptosis（P < 0.01）.3.Bioinformatics analysis showed that there were two binding sites between miR-29a-3p and 3’UTR of COL3A1.The results of dual luciferase reporter assay showed that when the wild-type reporter gene vector was co-transfected with miR-29a-3p mimic,the luciferase activity was significantly inhibited（P < 0.01）,while the mutant reporter gene vector containing pmir GLO-Mt-COL3A1 3’UTR 、 pmir GLO-M1t-COL3A1 3’UTR or pmir GLO-M2t-COL3A1 3’UTR was co-transfected with miR-29a-3p mimic,There was no significant difference in luciferase activity.Pearson correlation analysis in 28 keloid tissues showed that miR-29a-3p was significantly negatively correlated with the expression level of COL3A1.4.Compared with KFBs cells transfected with miR-29a-3p mimic,the proliferation activity of cells in miR-29a-3p mimic+pc DNA3.1-COL3A1 group was significantly increased and the apoptosis rate was decreased（P < 0.05）;Compared with KFBs cells transfected with miR-29a-3p mimic,There was no significant difference in the expression of miR-29a-3p in miR-29a-3p mimic+pc DNA3.1-COL3A1 group（P > 0.05）.Conclusions1.The expression of miR-29a-3p was significantly low in keloid tissue;2.miR-29a-3p negatively regulates the expression of COL3A1 in keloid fibroblasts;3.Overexpression of miR-29a-3p targeting COL3A1 inhibits the proliferation and promotes apoptosis of KFBs cells.