Establishment Of Keratinocyte-specific MiR-142 Transgenic Mice

Cutaneous wound healing is a complex and exactly-regulated process involving-inflammation, re-epithelialization and formation of granulation tissue and finallytissue remodeling一that overlap in time. In the last few years there were more andmore studies on miRNA functioning in skin development and hair folliclemorphogenesis. Mature miRNAs are approximately 21-24 nucleotides [nt] in lengthderived from endogenous stem-loop precursor transcripts which can bind to 3'-untranslated region (3'一UTR) of target mRNA to inhibit target gene expression eitherby degradating mRNA or suppressing translation. It has been reported that miRNAsare involved in various signaling pathways and play comprehensive and importantroles in a variety of biological processes, however few miRNAs are reported toinvolve in the process of cutaneous wound healing. The miR-142 gene, located onchromosome 17, is high expressed in spleen and thymus gland, and plays a greatrole in the process of hematopoiesis and inflammation. However, the function ofmiR-142 in skin development and wound repair were still remained largely unknown.We detected the expression profile of miR-142 in 9 different organs, and found that miR-142 was high expressed in thymus gland. MiR-142 was also expressed in skin and up-regulated in the wounded skin, which suggested that miR-142 might play a crucial role in cutaneous wound healing. To further understand the role of miR-142 in wound healing, we next explored the biological effect of miR-142 in HaCaT in vitro. Overexpression of miR-142 significantly suppressed cell proliferation. In addition, flow cytometric analysis revealed that miR-142 overexpression induced a reduction in the amount of cells at S phage. The result of scraching and transwell showed that overexpression of miR-142 in HaCaT could attenuate cell migration. To make it clear that how miR-142 affect cell proliferation and migration, bioinformatics analysis was performed to search for miR-142 targets. We also detected the expression of the candidated targets responsible for regulating cell proliferation, including Pten, Smad4, Smad2, cyclin Dl and cyclin E2, in miR-142 overexpressed HaCaT cells, and found that cyclin Dl was downregulated. To investigate the role of miR-142 in skin development and wound repair ininvo, we tried to construct keratinocyte-specific miR-142 transgenic mice. First wemade a construct that directed the over-expression of miR-142 in keratinocyte with apromoter of KS (6.3kb) which has been reported. Seven miR-142 transgenic micefounders were generated through fertilized eggs microinjection followed by embryotransplantation. The keratinocyte-specific over-expression of miR-142 was confirmedby Northern blot. HE staining showed us that the 6-weeks-old transgenic micedisplayed normal epidermis, hair follicle and dermis. Furtherly immunohistochemisty ofK14, marked keratinocyte in the basal layer, and involucrin, markedwell-differentiated keratinocyte above the basal layer, showed us that the differentlayers of transgenic mice epidermis were normal.To sum up, over-expression of miR-142 which dramaticly increased after skininjuries downregulated the expression of cyclin Dl to attenuate cell proliferation andalso suppressed cell migration in HaCaT, indicating that miR-142 may play a crucialrole in cutaneous wound healing. Then we estabilished keratinocyte-specific miR-142transgenic mice that might be an ideal experiment animal model for understanding thefunction miR-142 in cutaneous wound healing in vivo and the molecular mechanismsof cutaneous wound healing.