Differential Expression Of MiR-let7a In Hair Follicle Cycle Of Liaoning Cashmere Goats And Identification Of Its Targets

The cashmere goat is a breed of goats that produce cashmere wool，and it mainlyconsists Australian cashmere goats、Iraq cashmere goats、Liaoning cashmere goatsand Hexi cashmere goats,et al.in the world. In our country, there are10species inthe Cashmere goat breeds of genetic resources protection, including Tibet Plateaugoats, Liaoning cashmere goats, Lvliang black goats, Inner Mongolia cashmeregoats and so on. To improve the production of cashmere in China, more and moreexcellent Cashmere goats are cultivated. Moreover, Hair follicle plays a key role inimproving the quality of cashmere. Following the post-genomic era, there is a newpath to the growth and development of the hair follicle. In recent years, the researchof non-coding small RNA regulatory mechanisms has become a hot spot.MicroRNAs(miRNAs) constitute a large family of non-coding RNAs thatfunction as guide molecules in diverse gene silencing pathways, which involved inregulation of the growth and development of plants and animals, cell proliferationand differentiation, signal transduction and the immune response and so on. Sincethe first miRNA was discovered in1993by Victor Ambros and Rosalind Lee duringa study of the lin-4gene, which was known to control of C. elegans larvaldevelopment by repressing the lin-14gene, more and more reports indicated thatmiRNAs are well conserved in both plants and animals, and are thought to be a vitaland evolutionarily ancient component of genetic regulation. miRNA-let7family isone of the most in-depth studies on miRNAs, which plays an important role interms of cell proliferation,differentiation and tumor formation in animals. In recentyears, studies have indicated that let-7family can regulate the formation of skinmelanoma and expressed in the skin tissue of goats and sheep, but there is little research on its regulation of hair follicle cycle.In this study, the regulation mechanism of miRNA-let7a in hair follicle cyclewas studied in Cashmere goats. Firstly, quantitative PCR technique was used todetect differential expression of miRNA-let7a in anagen and catagen cashmeregoats, then bioinformatics software miRGenV3.0was used to predict the targetgenes of miRNA-let7a, and further to screen out the target genes related the hairfollicle cycle by DAVID6.7, including GO and KEGG pathway. Finally, thecandidate target genes of miRNA-let7a were identified by RT-PCR, Western-blottechnique and dual luciferase reporter. And transfection efficiency was optimized byMTT, fluorescence microscopy and flow cytometry. The study will clarify theregulation mechanism of miRNA-let7a in hair follicle cycle of Cashmere goats. Theresults as follows:(1).miRNA-let7a is expressed in the skin tissues of Cashmere goats in anagenand catagen, and the expression of miRNA-let7a in catagen was higher significantlythan that in anagen.(2). The miRGenV3.0software was used to predict target genes of miRNA-let7a,and three target genes were screened, Cmyc, FGF5and IGF-1R, which were relatedto hair follicle cycle. And homology analysis revealed that the homology ofmiRNA-let7a in Cashmere goats were higher than90%with ovis aries, bovine andother species.(3).The expression of Cmyc, FGF5and IGF-1R in anagen and catagen Cashmeregoats were detected from the molecular level and protein level by fluorescencequantitative PCR and Western-blot. The results showed that the expression of thethree target genes in anagen were higher than that in catagen from the molecularlevel, and the protein expression of Cmyc and FGF5were higher in anagen thanin catagen, and it was consistent with the quantitative results, opposite to theexpression of miRNA-let7a. The results showed that Cmyc and FGF5were thetarget genes of miRNA-let7a. (4).HEK-293T cells were transfected with liposomes, and detected by thefluorescence microscopy (FM), flow cytometry (FCM) and methylthiazoletrazolium(MTT). The results showed that:1μl added to each well at a final concentration ofliposomes, mimics co-transfected with a plasmid vector for100nM and800ngrespectively, the highest transfection efficiency can be reached53.13%.(5).Dual luciferase assay system was used to identify the binding site of targetgenes and miRNA-let7a. The results showed that the binding site of Cmyc andmiRNA-let7a was CTGCCTC, the target site of FGF5and miRNA-let7a wasACTCCAT.