Function And Mechanism Of MiR-29 And Its Target Gene COLI In Hypertrophic Scar Formation

Background:Hypertrophic scar(HS)as a common complication of skin wound healing process,with 4 ~16% incidence rate can seriously influenced organization function and appearance.The metabolism imbalance of collagen synthesis and degradation is the primary cause of the HS.DNA microarray showed that miRNA expression in normal skin and HS tissue exists significant difference,13 kinds of which are low expressed in HS,meanwhile 92 kinds of which are highly expressed in HS.These target genes regulated by miRNA involve cell proliferation and apoptosis,movement and cycle,metabolism and synthesis,etc.One of the most significant change of miRNA is miR-29 of which the change ratio is as high as 15.0.Pic Tar,Target Scan and miRanda bioinformatics analysis software revealed COLI is an effective target genes of miR-29.Therefore,we propose a continuous that persistent low expression of miR-29 lost effective regulation on COLI gene expression,resulting in of the excessive deposition of the collagen,eventually forming the HS.Therefore,increasing expression of miR-29 in hypertrophic scar may hopefully restrain the excessive fibrosis,and provide a promising target for the prevention and treatment of the HS.Objectives:In this study,by using a miRNA array-based analysis,we filtrated miRNA which is significantly differently expressed in hypertrophic scar tissue and normal skin tissues.We explore the mechanism of miRNA-29 regulating COLI in hypertrophic scar formation,so as to provide new targets for the biological treatment of the HS.Methods:1.Collected specimens of hypertrophic scar and normal skin tissue were verified by HE staining to prove whether these clinical specimens can meet experiment requirement.The original generation of cultured fibroblasts was derived from the hypertrophic scar(HS)and normal skin(NS).In this research,we employed the miRNA PCR array data analysis to explore which miRNA is differentially expressed in HS and NS.2.The expression level of miR-29 c and COLI in HS and NS was detected by RT-PCR,and was Further validated by WB.The correlation of expression between miR-29 and COLI was analyzed by chi-square test or Fisher’s exact test;3.We employed a dual-luciferase reporter system,which was used by co-transfection of miR-29 c and a luciferase reporter plasmid containing the 3’ UTR of human COLI,to verify whether COLI is a direct target of miR-29 c.4.We builded miR-29 overexpressed adenovirus and then respectively transfected adenovirus into fibroblasts.By using flow cytometry and CCK 8,we explore the function and mechanism of miR-29 c in hypertrophic scar.Results:1.Collected specimens of normal skin and hypertrophic scar tissue can meet experiment requirement.Immunofluorescence staining confirmed that the original generation of cultured cells derived from hypertrophic scar tissue and normal skin tissue were fibroblasts.2.Gene chip technology was used to screen miRNAs that was differently expressed in normal skin and hypertrophic scar tissues.The result showes that the expression of 45 miRNAs were upregulated in hypertrophic scars,comparing to norma skin tissue,such as miR-143、miR-32、miR-92 b 及 miR-25.Wheras,there are 68 miRNAs are downregulted,such as miR-29a、miR-29b、miR-29c、miR-713、miR-195.Among them,the change of miR-29 is the most significant in hypertrophic scar tissues than in normal skin.3.Low miR-29 c expression was observed in significantly more hypertrophic scar tissue samples compared with normal skin tissue samples,as well as in the fibroblasts.4.COLI was forcasted to be the target gene of miR-29 by bioinformation softwares,and it was highly expressed in hypertrophic scar tissue samples compared with normal skin tissue samples.The correlation of expression between miR-29 and COLI was negatively correlated;5.We used the dual-luciferase reporter system to verify whether COLI is a direct target of miR-29.Mi R-29 c minic can significantly inhibit the luciferase activity of 3’ UTR of human COLI,however miR-29 c minic failed to inhibit the luciferase activity of 3’ UTR of human COLI mutant,suggesting that miR-29 c can directly target COLI.6.Moreover,in fibroblasts cells,endogenous expression of COLI protein was significantly suppressed by miR-29 c transfection,further proving that miR-29 c expression can directly negatively regulated COLI.Cell proliferation experiment and flow cytometry analysis found that proliferation rate of fibroblasts with miR-29 c overexpression increased significantly and inversely its apoptosis rate dropped significantly.Conclusions:1.Gene screen shows that the expression of 45 miRNAs were upregulated in hypertrophic scars,comparing to norma skin tissue,such as miR-143、miR-32、miR-92b及 miR-25.Wheras,there are 68 miRNAs are downregulted,such as miR-29a、miR-29b、miR-29c、miR-713、miR-195.Among them,the change of miR-29 is the most significant in hypertrophic scar tissues than in normal skin.2.miR-29 c was significantly highly expressed in normal skin tissue than in hypertrophic scar tissue,Lower expression of miR-29 c in the hypertrophic scar can promote the proliferation and inhibit the apoptosis of fibroblasts.All these results suggested that miR-29 c could be negative regulator in the formation of hypertrophic scars.3.As COLI genes is miR-29 c directly target genes,miR-29 plays an important role in the HS through the negative regulating COLI gene expression.So miR-29 c is likely to become the new targets for the HS treatment.