Screening Of Hair Follicle Related MiRNAs And Analysis Of Target Gene Expression In Fgf21 Knock Out Mice

Fibroblast growth factor 21(Fgf21)is a factor closely related to the study of hair follicles,which plays a key role in the growth cycle of hair follicles.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs,which have been found to regulate the growth and development of mammalian hair follicles.In this study,we sequenced the back skin tissue of wild type(WT)and Fgf21 knockout(KO)mice by high-throughput miRNA sequencing technology,and screened out 10 differentially expressed miRNAs related to hair follicles.It is speculated that miRNAs and their target genes may participate in the regulation process of Fgf21 on hair follicle growth and development.In order to explore the effects of Fgf21,miRNA and their target genes on hair follicles.Real-time PCR,Western blot,HE staining,immunofluorescence,dual-luciferase reporter gene detection system and bioinformatics analysis were used to explore the interaction between the three andtheir effects on hair follicle development.1.Bioinformatics analysisBioinformatics analysis showed that 10 miRNAs hair follicle related target genes got five functional attributes in GO functional annotation: cytoplasm,nucleus,protein binding,identical protein binding,positive regulation of transcription from RNA polymerase II promoter.Combined with literature reports about genes that regulate the growth and development of hair follicles,we finally screened and mapped out the miRNA-GO-target gene network association diagram of 12 target genes(Tgfbr2,Wnt7 a,Snap29,Abcg2,Map3k1,Hcn1,Ccnd2,Notch3,Bcl2,Vezf1,Trps1,Antxr1).In KEGG signaling pathway annotation,seven signaling pathways were obtained: Wnt signaling pathway,SNARE interactions in vascular transport,ABC transporters,protein processing in endothelial reticulum,Notch signaling pathway,ubiquitin mediated protocol and MAPK signaling pathway.Combined with literature reports and related genes and signal pathways involved in regulating the growth and development of hair follicles,we finally screened and drew the miRNA-KEGG-target gene network association diagram of 7 target genes(Tgfbr2,Wnt7 a,Snap29,Abcg2,Map3k1,Notch3,Bcl2).2.Prediction and validation of miR-134-5p and miR-136-5p target genesIn order to study the mechanism of action of miR-134-5p and miR-136-5p,bioinformatics software was used to predict the target genes,and then double luciferase reporter gene detection system was used to test.Finally,we found that Vezf1 was the target gene of miR-134-5p,and Map3k1 was the target gene ofmiR-136-5p.3.Effect of Fgf21 gene on the expression of Vezf1 and Map3k1In the first natural growth cycle of hair follicles,the results of Real-time PCR and Western blot showed that there was no significant difference in the expression of Vezf1 and Map3k1 in the hair follicle anagen of WT and KO mice(P > 0.05);In the hair follicle catagen,the expression of both in the back skin of KO mice was higher than that of WT mice(P < 0.005);in the telogen of hair follicles,the expression of both in the back skin of KO mice were lower than that of WT mice(P < 0.005).The results of immunofluorescence showed that the target genes Vezf1 and Map3k1 were affected by Fgf21 gene,and the secretory sites of proteins also changed in different degrees.Vezf1 and Map3k1 were secreted in different degrees in the dermal papilla,hair bulb,outer root sheath and inner root sheath of hair follicles,while the dermal papilla,hair bulb,outer root sheath and inner root sheath of hair follicles were also the main components of mouse hair follicle structure.Therefore,Vezf1 and Map3k1 play an important role in regulating the growth and development of mouse hair follicles and hair follicle growth cycle.