The Regulation Of Hair Follicle Development By MIR-148B And MIR-320in Sheep

MicroRNAs (miRNAs) are a class of tiny non-coding RNA which can widely regulate the expression of most genes s in eukaryotes. It was found to participate in tumorigenesis, hart development, fat metabolism, neurogenesis, cell proliferation and differentiation and so on. At the same time, it is involved in hair follicle development as well. In order to identify the key miRNAs in hair follicle development,our previous study had used solexa sequencing to detect the miRNAs expression profiling of hair follicle from different stage(Telogen,Anagen,Catagen) for Tebitan sheep.Among these differntial expressed miRNAs,miR-148b and miR-320were highly-regulated from Telogen to Anagen.On the other hand,there is not enough evidence to suggest that miR-148b or miR-320can affect the growth of hair follicle.HHDPC and HHGMC were used as materialsinvitro to study the role of miR-148b and miR-320in these cells’proliferation in the development of the hair follicle in this study,and indentify the novel targets of miR-148b during this procedure.The results we finally got were stated as follows:The expression of miR-148b and miR-320were increased from telogen to anagen in the developmen of hair follicle.Q-pcr was also used to detect the expression of miRNAs in hair follicle from different stage,which in consistent with the sequencing result of the early.MiR-148b can promote the proliferation of HHDPC and HHGMC,while miR-320will repress their proliferation.HHDPC and HHGMC were transfected with miR-148b and miR-320,and subsequently the prolifetating cells were stained by Edu.It was turn out that the Edu-positive cells in miR-148b over-expressed group were much more than in control group(P<0.05),while the Edu-positive cells in miR-320over-expressed group were less more than in control group(P<0.05).The cell growth dynamics monitored by real-time xCELLigence system showed that miR-148b transfected HHDPC cells bebaving a lagging growth compared to control cells.miR-148b can significantly improve the activity of Wnt signaling pathway. Cotransfect TOPFlash/FOPFlash and miRNAs to293T cells and found that miR-148b can significantly increase the fluorescence signal intensity of TOPFlash (P<0.05).miR-148b can significantly increase the expression of some key genes of Wntsignaling pathway,while miR-320can significantl decrease their expresion.The expression of β-catenin、c-myc、cycD、c-jun and PPARD were detected in miR-148b or miR-320transfected HHDPC cells.The results showed that miR-148b can increase the expression of these genes while miR-320can decrease their expression.MiR-148b directly target SMAD2,CAMK2A,NFAT5and WntlOb by binding their3’UTR. After Targetscan predicting and screening, NFAT5,SMAD2, CAMK2A, PRICKL2, Wntl and Wnt10B were chosen to be the candidate targets of miR-148b.The specifict binding of miR-148b and SMAD2, CAMK2A, NFAT5orWntlOb3’UTR were confirmed by Dual-Luciferase reporter system.