Molecular Mechanism Of MiR-199a-5p Regulating Dermal Papilla Cells Proliferation Of Cashmere Goat

The development of hair follicle has the characteristic of periodic development,which includes three stages:anagen、telogen and catagen and the dermal papilla cells are the key signal and tissue center of hair follicle development.miRNA significantly regulates the expression of related genes during the hair follicle growth and development of cashmere.Bioinformatics analysis of miRNA sequencing results of cashmere skin tissues in the early stage of the research group showed that miR-199a-5p expression level was significantly higher in telogen than in anagen and miR-199a-5p has been reported to inhibit the proliferation of many cells such as skin keratinocytes and vascular endothelial cells.However,its role in dermal papilla cell proliferation of cashmere hair follicle is still unknown.Therefore,in this study,the dermal papilla cells of cashmere hair follicle were taken as the object.MTT and Ed U assays were used to detect the proliferation of dermal papilla cell by overexpression/inhibition miR-199a-5p and the expression of proliferationrelated genes was further verified and then to study its regulatory mechanisms,the following main results were obtained:1.MTT and EdU were used to study the function of miR-199a-5p in dermal papilla cells.MTT results showed that the cell viability of dermal papilla cells after overexpression of miR-199a-5p was significantly reduced(P < 0.01).However,the survival rate was significantly increased after inhibition of miR-199a-5p(P < 0.01);After Ed U staining,which showed that the number of positive cells in the miR-199a-5p overexpression group was significantly lower than that in the control group,while the number of positive cells was significantly increased after inhibition of miR-199a-5p expression compared with the control group.The expression quantity of proliferation-related genes PCNA and C-Myc also lower than that in the control group after overexpression of miR-199a-5p which indicated that miR-199a-5p could inhibit the proliferation of dermal papilla cells.2.Studies on the targeting relationship between miR-199a-5p and VEGFA indicated that the ratio was significantly lower than that of the control group after overexpression of miR-199a-5p and significantly higher than that of the control group after inhibition of miR-199a-5p.However,there was no significant difference in the relative activity ratio of VEGFA after mutating the targeted binding site,suggesting that miR-199a-5p regulates VEGFA expression by targeting VEGFA 3’UTR region.q RT-PCR,western blot assay and immunofluorescence assay further verified that VEGFA was the target gene of miR-199a-5p.3.Building adenovirus to overexpress VEGFA and co-transferred to cashmere goat with miR-199a-5p mimics to rescue experiment,Results showed that VEGFA promoted the proliferation of dermal papilla cells in cashmere,which was contrary to miR-199a-5p.It further indicates that miR-199a-5p inhibits the proliferation of dermal papilla cells of cashmere by inhibiting the expression of VEGFA gene.This study revealed the role of miR-199a-5p in the proliferation of dermal papilla cells in cashmere and its regulatory mechanism,providing a theoretical basis for revealing the molecular mechanism of hair follicle development in cashmere.