A Study On The Effects Of Improving Skin Wound Healing By Transgenic Therapy Of MiR-21

Wound-healing is one of the important subjects in modern medicine. Studies of thewhole process of cutaneous wound healing at the molecular level have promoted theresearch on wound healing to an unprecedented level. A series of researchs on growthfactors, signaling pathways and functional proteins have deepen our understanding ofmechanism of wound healing,as well as provided many clues on treatment and medicine. Inrecent years, the important function of microRNAs has revealed in many biological events,meanwhile its function during cutaneous wound healing has been paid more attention.Researchs from both others and us have shown that the oncogene miR-21was upregulatedafter skin injury, and inhibition of miR-21in the local wounds resulted in delayed woundhealing, which indicated that miR-21is one factor to promote wound healing and onepotential therapeutic molecular target of wound healing. However, others reported thatmiR-21was also upregulated in samples of venous ulcers, and wound healing delayed bylocal injection of miR-21mimics in rat model. These contradictory results make us askwhich kind of effects would be when miR-21overexpressed in wounds and whether miR-21will be one potential therapeutic target to promote wound healing. To clarify these questions,the main methods and results are as followed:1． Construction of the eukaryotic expression vector pRc/CMV-miR-21andidentification of its biological activity. One382bp-length DNA sequence containingpre-miR-21flanking with Hind Ⅲ and XbaⅠ ebdonuclease sites, obtained by PCR usingmouse genomic DNA as the template, was inserted into multiple clone sites of plasmidpRc./CMV. The synthetic oligonucleotides containing reverse complement sequence tomature miR-21flanking with Sac I and Hind Ⅲ ebdonuclease sites, was inserted intomultiple clone sites in the3′UTR of luciferase gene of plasmid pMIR-Report. Expression ofmature miR-21of pRc/CMV-miR-21was demonstrated by Northern blot in stabletransfected293cells and its function was identified by luciferase assay.2．Establishment of in vivo transgenic therapy mouse wound healing model by local subcutaneous injection of pEGFP-N1reporter plasmid. Mice were anesthetized and circularfull-thickness skin excisions of10mm in diameter in the middle of back were generated.The2oo ul naked pEGFP-N1plasmid with200ug/ml was injected locally with three sitesto the wounds respectively. GFP immunohistochemistry was stained in sections of varioustimepoints. GFP positive cells were predominantly present in the granulation tissue.And thepeake time of GFP reporter gene expression was7-10thdays, while in the15thday itsexpression reduced dramaticly. Exogenous miR-21was present in the wound detected by insitu hybridization of miR-21by the pRc/CMV-miR-21plasmid.3．Local subcutaneous injection of naked plasmid miR-21into the mouse woundhealing models significantly improved wound healing. Wound healing models of nomarlmice and type2diabetes mice were made and pRc/CMV-miR-21naked plasmid wasinjected as well as its control empty plasmid. Mean wound healing time and residual woundareas was determined. HE and masson staining were used to reflect wound histopatholgicalchanges during wound healing. Mean wound healing time of miR-21transgenic group was11d, which was shorter comparing with control group (13d). The result of HE stainingshowed that the miR-21transgenic group was superior to the control group featured bymore migratory fibroblasts and keratinocytes. As miR-21is one pro-fibrogenic factor, thedeposition and reformation of collagen in30d wounds were evaluated by Masson staining,which showed no difference between the two groups.