Effects Of MiR-31-5p Targeting RASA1 Upregulating MAP3K1 Of Hair Follicle Stem Cells In The Yangtze River Delta White Goat

The Yangtze River Delta White Goat is the only goat species discovered so far that can produce superior-quality brush hair(Type Ⅲ).The transcriptome sequencing results of the research team showed that there are significant differences in the expression level of MAP3K1 in the individual skin tissues of superior-quality brush hair and normal-quality brush hair.This study aims to explore the key miRNAs that target with MAP3K1 during the formation of superior-quality brush hair and their effects on the proliferation and apoptosis of goat hair follicle stem cells.The bioinformatics websites(StrBase,miRDB,TargetScan and miRWalk)were used to predict and select the miRNAs,with targeted relationship with MAP3K1,and use the online website Venny 2.1 to draw a Venn diagram.Through the construction of miR-31-5p overexpression vector and miR-31-5p RNA oligos,MAP3K1,RASA1 Wild/Mutant-type Dual-Luciferase Reporter assay vector,the relationship between miR-31-5p and MAP3K1,RASA1 was verified.qPCR and Western Blot assays further used to detect the effect of overexpression or inhibition of miR-31-5p on MAP3K1,RASA1 mRNA and protein expression levels.In order to explore the effects of miR-31-5p on cell proliferation and apoptosis,the proliferation-related genes(PCNA,CDK1,CCND2)and anti-apoptotic gene(Bcl-2)in hair follicle stem cells after overexpression or inhibition of miR-31-5p were analyzed.And pro-apoptotic gene(Bax)mRNA and protein expression levels;cell cycle and cell apoptosis of hair follicle stem cells were verified by CCK-8,EdU,flow cytometry.The results of this research are as follows(1)Three miRNAs that may interact with MAP3K1 were jointly predicted through the database.The final selection scores were relatively high,combined with previous reported study in miRNAs on skin and hair follicle cells,and miR-31-5p was selected as the research object.(2)miR-31-5p is expressed in various tissues of Yangtze River Delta white goats,and the expression level of miR-31-5p in the skin tissues of individuals with superior-quality brush hair in Yangtze River Delta white goats is higher than that in individuals with Normal-quality brush hair.(3)Overexpressing or inhibiting miR-31-5p in the hair follicle stem cells in the Yangtze River Delta White Goat,and detecting its effect on the expression level of proliferation and apoptosis-related genes by qPCR and Western Blot;Through CCK-8 and EdU experiments to explore the effect of miR-31-5p on cell proliferation;combined with flow cytometry to detect the effect of miR-31-5p on cell cycle and cell apoptosis.The results confirm that miR-31-5p promotes the proliferation of hair follicle stem cells and inhibits apoptosis.(4)Successfully constructed MAP3K1,RASA1 Dul-Luciferase Reporter assay vector.The results of the Dul-Luciferase Reporter assay indicated that miR-31-5p up-regulated the expression level of MAP3K1 by targeting the upstream inhibitor RASA1 in the MAPK signal pathway.qPCR and Western Blot confirmed that miR-31-5p inhibited the expression of RASA1 and increased the level of MAP3K1.This study reveals the mechanism by which miR-31-5p regulates the proliferation of hair follicle stem cells,that is,miR-31-5p participates in the MAPK signaling pathway to target the inhibition of RASA1 and up-regulate the expression level of MAP3K1,thereby promoting the proliferation of hair follicle stem cells and inhibiting apoptosis.This experiment provides a theoretical basis for further elucidating the molecular formation mechanism of superior-quality brush hair traits in the Yangtze River Delta White Goats.