| The hair follicles (HFs) are subsidiary organ of the skin, including20types of cells. Inmammals, hair follicles are divided into two classes for epithelial cells and dermal cells, thecyclical variation of hair follicle depends on the interaction between the dermal papilla andthe follicular epithelium. Cashmere goat hair follicles comprise primary and secondaryfollicles, which independently generate the fiber products of wool and cashmere, resulting indifferent economic values due to the differences in the fibers’ diameters. Cyclical changes inthe primary follicle are not obvious, but the secondary follicles undergo periodical cycles in ayear through anagen, catagen and telogen stages. MiRNAs play important roles in the growthand development, organogenesis, cell differentiation, proliferation and apoptosis, cell cycleconversion, muscle and fat metabolism in mammals. Accumulative evidences show that theexpression of skin miRNAs changes remarkably during distinct stages of the hair cycle inhumans, mice, goats and sheep. In this study, the use of high-throughput sequencing, cellculture, cell transfection, experimental techniques, gene cloning and gene expression analysiscombined with bioinformatics. Shanbei Cashmere goat secondary follicle development relatedmiRNA periodic screening and identification, and the candidate for the preliminary functionalmiRNA research. The main results are as follow:1. The skin tissues were harvested from the three stages of hair follicle cycling (anagen,catagen and telogen) in a fiber-producing goat breed. In total,63,109,004raw reads wereobtained by Solexa sequencing and15,997,828、18,111,912and27,016,012clean readsremained for the small RNA digitalization analysis. Among these sequences, the highestpercentages of these sRNAs were22-nt long, which is consistent with the common size ofmiRNAs.2. We were identified399conserved miRNA of secondary hair follicle cycling ofcashmere goats. In addition, the length of the miRNA sequences ranged22nt and their5’ endswere comprised most frequently of uridine (U). Among these,326miRNAs were expressed inall three follicular cycling stages, whereas3,12and11miRNAs were specifically expressedin anagen, catagen, and telogen, respectively. We also identified172potential novel miRNAs by Mireap,36miRNAs were expressed in all three cycling stages, whereas23,29and44miRNAs were specifically expressed in anagen, catagen, and telogen, respectively.3. GO enrichment analysis for target genes based on biological process showed that9,981genes were related to biological processes.79.4%of the genes were involved in cellularprocesses, and55.8%and47.2%of the genes were involved in metabolic processes andbiological regulation, respectively. KEGG pathway annotation showed that10,563targetgenes were annotated for278biological functions. Most of these genes were involved incellular metabolism, diseases and signal transduction. The most commonly indicated pathwaywas the Metabolic pathways, with1,212genes representing11.49%of the total target genes,followed by MAPK signaling pathway (3.01%), Endocytosis (2.68%), Focal adhesion (2.66%)and Regulation of actin cytoskeleton (2.45%).4. miR-125b was selected for further analyses because its expression in the anagen andcatagen, telogen stages was significant different. By evaluating the expression patterns ofmiR-125b in multiple tissues of cashmere goats, miR-125b was highly expressed in skin andbrain, and lowest expression level was found in liver.5. The Prediction of miR-125b target genes using TargetScan software obtained2881target genes. Compared to the previously reported genes involved in the hair follicle signalingpathways (Wnt, TGF-β, Notch, SHH, MAPK and Jak-STAT) and the differentially expressedgenes in the three hair cycling stages, five genes (FGF1, FGF5, TGF-β1, TGF-β3, TNF-α)were remained for further analysis. The FGF1gene was further removed due to mutatedsequences by amplifying this gene in both goat and cattle. The FGF5, TGF-β1, TGF-β3andTNF-α gene were selected for dual luciferase reporter gene assay validation, the resultsshowed miR-125b mimics can inhibit the wild-type of FGF5, TGF-β3and TNF-α. However,by binding to a mutated sequence of these four genes, only the FGF5luciferase activity canbe restored. Thus, miR-125b can adjust the predicted target FGF5target site.5. Observed agglutination growth by primary and secondary follicle dermal papilla cellsof Cashmere goat hair follicles in vitro, detected by immunofluorescence positive expressionof α-SMA, Versican and CD133, indicating a successful culture approach for goat primaryand secondary follicle dermal papilla cells.6. Pri-miR-125b was amplified from DNA of Cashmere goat. Recombinant plasmidpAd-miR-125b was constructed using AdEasy-1system. Enzyme digestion analysisdemonstrated that the recombinant adenovirus was constructed successfully. The virus waspackaged and amplified in293cells. Virus titer was identified by TCD50assay, the virus titerreached of2.30×109pfu/mL of pAd-miR-125b, and the empty viral titer reached of2.05×109pfu/mL of pAd-GFP. The expression of miR-125b and13genes related to secondary follicle dermal papilla cells of Cashmere goats were analyzed by qRT-PCR. The results of qRT-PCRshowed that the expression of miR-125b in cell infected by pAd-miR-125b (MOI=300) was2.6times than that in the control group. The mRNA expression of FGF5(p <0.01), IGF-1(p<0.01), Shh (p <0.01), TNF-α (p <0.01), Msx2(p <0.01), Lef-1(p <0.01), FGF7(p <0.05),β-catenin (p <0.01) genes were down-regulated and BMP2mRNA were up-regulated, themRNA expression of BMP4, Noggin, TGF-β1and BDNF were changed insignificant. Takentogether, miR-125b plays a regulatory role in hair cycling by inhibiting the expression of theFGF5gene related to cyclical changes in the hair follicles.During the anagen-catagen-telogen transformation of the hair cycle in cashmere goats,399conserved miRNAs and172novel miRNAs were found via ahigh-throughput sequencingapproach. Our findings enrich the caprine miRNA databases and provide new insights into themiRNA transcriptome in Cashmere goat skin and the hair follicle cycle; miR-125boverexpression by Ad-miR-125b could affect gene mRNA level related to secondary hairfollicle cycling in Cashmere goat secondary follicle dermal papilla cells. |
PDF Full Text Request