Involvement Of MiRNA-21 Is The Regulation Of Hair Follicle Development Cycle In Cashmere Goats By Targeting FGF18 And SMAD7

Cashmere goats have become a popular breed for farmers because of their superior cashmere and high cashmere yield.The cashmere of Cashmere goats is determined by the number of hair follicles and the state of hair follicles.Hair follicles can be divided into primary hair follicles and secondary hair follicles,and high quality cashmere is mainly produced by secondary hair follicles.In order to improve the economic value of Cashmere goats,increasing the yield of cashmere has become a new pursuit direction in animal husbandry industry.However,the traditional way to increase cashmere yield is mainly through hybridization or breeding excellent varieties,which is time-consuming,costly and unstable.With the progress of science and technology,it has become a new research hotspot to regulate the development cycle of hair follicles at the gene level and improve cashmere yield.The cycle of hair follicle development is from anagen to catagen to telogen.Regulating the periodic development of hair follicles,accelerating into anagen or delaying the end of anagen will effectively increase the output of cashmere.The development cycle of hair follicles is regulated by many biomolecules and signaling pathways,including miRNA.miRNAs a short sequence noncoding RNA molecules that are expressed in a wide range of eukaryotes,and miRNAs are important regulators of many cellular processes.miRNAs regulate gene expression by binding to incomplete complementary sites in 3’-UTRs of target m RNAs and participate in a variety of biological functions.Many studies have shown that miRNA can play an important part in hair follicle development,and multiple miRNAs were differentially expressed in different development stages of hair follicles through sequencing,including miRNA-21,which found by my groups previously.However,the mechanism of miRNA-21 regulating the development cycle of hair follicles is still unclear.Therefore,miRNA-21 was selected as the research object in this study.Target genes related to hair follicle development were predicted and screened online with Target Scan,miRBase and other software.To detect the expressions of miRNA-21 and its target genes in skin tissues of Cashmere goats at different developmental stages in vitro and in vivo,identify the targeting relationship between miRNA-21 and it’s target genes.To preliminarily clarify the role of miRNA-21 in the development cycle of hair follicles,and provide a theoretical basis for improving the yield of cashmere in the future.In this study,the expression of miRNA-21 in the skin tissue of Cashmere goats at telogen and anagen was detected.The results of RT-PCR showed that the expression of miRNA-21 in the skin tissue of Cashmere goats at telogen was significantly higher than that in the skin tissue of Cashmere goats at anagen,suggesting that miRNA-21 may be involved in the regulation of hair follicle cycle.The target genes of miRNA-21 were predicted by Target Scan and other online biological software,and the target genes related to hair follicle development were screened by GO and KEGG analysis software in DAVID.FGF18 and SMAD7 were identified as the target genes of miRNA-21.The m RNA and protein expression levels of FGF18 and SMAD7 were identified by RT-PCR and Western blot.The results showed that the expression of target genes FGF18 and SMAD7 in the skin tissue of Cashmere goats at telogen was significantly higher than that at anagen,and the expression trend was consistent with miRNA-21.To further identify the targeting relationship between miRNA-21 and target genes FGF18 and SMAD7.Dual-luciferase assay was performed to identify specific binding sites by constructing wild-type and mutant plasmids.The results showed that FGF18 and SMAD7 were the target genes of miRNA-21,and the binding site was AUAAGCUA.Finally,in order to further study the regulatory mechanism of miRNA-21.Ha Ca T cells were transfected with miRNA-21 mimics /inhibitor to increase and decrease the level of miRNA-21 in Ha Ca T cells.Then,the differential expression of miRNA-21 in different treatment groups was detected after RNA extraction.The results showed that the expression of miRNA-21 in the mimics group was significantly increased.While the results in the inhibitor group were contrary,indicating that the transfection was successful.Then the m RNA and protein levels of target genes were identified.Both results showed that the expression of target genes FGF18 and SMAD7 in the mimics group was significantly increased.While the expression of target genes in the inhibitor group was decreased,which was consistent with the expression trend of miRNA-21.These results indicated that miRNA-21 was involved in hair follicle development by positively regulating target genes FGF18 and SMAD7.It was found that miRNA-21 could participate in the regulation of hair follicle development cycle of Cashmere goats by positively regulating the expression of target genes FGF18 and SMAD7 by this experiment.And this experiment can be used as a new way to improve cashmere yield.