| As an excellent local breed of cashmere goat in China,the Inner Mongolia cashmere goat is known for its high quality and high yield of cashmere production,and cashmere is a high-grade textile material with high economic value.The growth and development of cashmere is affected by seasonal light.melatonin,an indoleamine secreted by the pineal gland,whose secretion is regulated by light and the environment,can induce cashmere growth by increasing the density of secondary hair follicles in the cashmere goat.mircoRNA(miRNA)is a class of short-chain non-coding RNA molecules,which inhibits the translation of mRNAs by targeting and binding to the 3’UTR region of mRNAs and plays a key regulatory role in the development and growth of the hair follicle.and growth,but the melatonin-mediated regulation of miRNAs on hair follicle growth is unknown.In this study,in order to investigate the melatoninmediated regulation of key miRNAs on hair papilla cells in velvet goats,we initially sequenced the whole transcriptome of the skin of Inner Mongolian velvet goats in the control and melatonin-embedded groups during the early growth period(May),and screened out the key miRNAs that were significantly correlated with the hair follicle growth and development(chi let-7d-5p and chi-miR-1388-3p)by bioinformatics methods,and utilized chi-miR-1388-3p and chi-miR-1388-3p to identify the key miRNAs that were significantly correlated with the hair follicle growth and development.1388-3p),and utilized dual luciferase reporter assay,qRT-PCR,Western Blot,flow cytometry and other techniques to explore the melatonin-mediated regulation of key miRNAs on the phenotype of hair papilla cells as well as the molecular mechanism in velvet goats.The main findings are as follows:1.Construction and sequencing analysis of mRNA and miRNA libraries of melatonin-regulated Inner Mongolia velvet goat skinSix mRNA libraries and four miRNA libraries of melatonin-embedded and control groups of Inner Mongolia downy goat skin in the early growth period were constructed by RNA-Seq technology.Based on bioinformatics analysis,a total of 244 differentially expressed mRNAs were identified,including 113 up-regulated mRNAs and 131 downregulated mRNAs,which were involved in the regulation of the cell cycle,organelle parts,cellular ECM-receptor interactions,Notch signaling pathway,etc.The miRNA sequencing analyses revealed that the mRNA libraries of melatonin in the skin of Inner Mongolia downy goat buried group and control group in the early growth period were identified as the most important mRNA libraries;miRNA sequencing analysis identified 170 miRNAs that were differentially expressed between the two groups of samples,of which 67 were up-regulated miRNAs and 103 were down-regulated miRNAs,and 2 key candidate miRNAs were screened out,namely,chi let-7d-5p and chi-miR-1388-3p.Functional enrichment analysis of miRNA target genes showed that the target Functional enrichment analysis of miRNA target genes showed that the target genes were related to transmembrane transport,protein modification,and MAPK,Notch,Wnt and Ras signaling pathways.The mRNA and miRNA regulatory network revealed the relationship between the differentially expressed miRNA and its target genes in the hair follicles of cashmere goats under the regulation of melatonin.2.Effects of melatonin on the phenotype of hair papilla cells in Inner Mongolian goatsFirstly,the hair papilla cells of Inner Mongolia velvet goat were successfully isolated and cultured by enzyme digestion method,and the expression of α-SMA and VIM,which are markers of hair papilla cells,was confirmed by immunofluorescence staining;using CCK8 method and flow cytometry,it was found that melatonin could promote the proliferation of hair papilla cells,increase the ratio of the cells in the Sphase to those in the G2/M-phase,and inhibit the apoptosis of cells;the results of qRTPCR and Western Blot showed that melatonin could significantly promote the proliferation marker gene Ki67 and the proliferation marker gene VIM in the hair papilla cells.PCR and Western Blot results showed that melatonin could significantly promote the expression of proliferation marker genes Ki67 and PCNA in hairy papilla cells at both mRNA and protein levels(P<0.01).3.Melatonin mediates the effect of chi-let-7d-5p on the phenotype of hair papilla cellsInterference and overexpression of chi-let-7d-5p in hair papilla cells revealed that the interference group was able to significantly promote the proliferation of hair papilla cells,significantly inhibit apoptosis,and increase the ratio of cells in the S phase to those in the G2/M phase(P<0.01),whereas overexpression of chi-let-7d-5p significantly inhibited the proliferation of hair papilla cells,promoted apoptosis,and significantly decreased the ratio of cells in the S phase to those in the G2/M phase cell ratio(P<0.01);addition of melatonin to the cell lines of the overexpression group revealed that melatonin could mediate the down-regulation of chi-let-7d-5p to significantly promote the proliferation of hairy papilla cells,promote the cell ratio of cellular S-phase to G2/M-phase,and significantly inhibit the apoptosis of cells(P<0.01).4.Melatonin mediates the molecular mechanism of chi-let-7d-5p in hair papilla cellsThe dual-luciferase reporter assay showed that chi-let-7d-5p was able to bind to the predicted target site of WNT2.qRT-PCR and Western Blot showed that chi-let-7d5p targeted the 3’UTR of WNT2 and significantly inhibited its mRNA and protein expression(P<0.01),and negatively regulated the Wnt signaling pathway related gene mRNA and protein expression(P<0.01),as well as negatively regulated the Wnt signaling pathway related genes WNT2 and WNT2 related genes.The results of Western Blot showed that chi-let-7d-5p targeted the 3’UTR of WNT2 and significantly inhibited its mRNA and protein expression(P<0.01),and negatively regulated the expression of Wnt10B and β-catenin mRNA and protein,which are related to the Wnt signaling pathway;Melatonin could mediate the down-regulation of chi-let-7d-5p and significantly promoted the expression of Ki67,a marker gene for hair papilla cell proliferation,and PCNA,as well as the expression of WNT2 and its related genes of the Wnt signaling pathway(P<0.01).5.Melatonin-mediated effects of chi-miR-1388-3p on hair papilla cell phenotypesInterference and overexpression of chi-miR-1388-3p in hair papilla cells revealed that interference with chi-miR-1388-3p significantly promoted proliferation and inhibited apoptosis of hair papilla cells,and significantly increased the ratio of cells in the S phase of the cell to those in the G2/M phase of the cell(P<0.01),while overexpression of chi-miR-1388-3p significantly inhibited proliferation of hair papilla cells,promotes apoptosis and significantly reduces the cell ratio of cell S phase to G2/M phase(P<0.01);addition of melatonin to the cell lines of the overexpression group revealed that melatonin could mediate the down-regulation of chi-miR-1388-3p to significantly promote the proliferation of hairy papillae cells,promote the cell ratio of cell S phase to G2/M phase,and significantly inhibit the apoptosis of cells(P<0.01).6.Melatonin mediates the molecular mechanism of chi-miR-1388-3p in hair papilla cellsThe dual-luciferase reporter assay showed that chi-miR-1388-3p was able to target the 3’UTR of PDGFA.qRT-PCR and Western Blot showed that chi-miR-1388-3p targeted PDGFA and significantly inhibited the expression of its mRNA and protein(P<0.01),and negatively regulated the MAPK signaling pathway related gene MAPK(P<0.01),as well as negatively regulated the MAPK signaling pathway related gene MAPK(P<0.01),and negatively regulated the MAPK signaling pathway related gene MAPK.The results showed that chi-miR-1388-3p targeted PDGFA and significantly inhibited its mRNA and protein expression(P<0.01),and negatively regulated the expression of MAP3K1 and FGF21 mRNA and protein,which are related genes of MAPK signaling pathway;melatonin mediated the significant inhibition of chi-miR1388-3p on the expression of Ki67,PCNA,and PDGFA,which are markers for proliferation of hairy papillary cells,as well as the expression of PDGFA and its related genes of the MAPK signaling pathway(P<0.01).In summary,in this study,we constructed an mRNA and miRNA library of melatonin-regulated skin tissues in the early stage of growth in Inner Mongolia velvet goat,screened and obtained two key miRNAs and signaling pathways in melatoninregulated hair follicle growth in velvet goat,and mapped the miRNA-mRNA interactions network.The systematic analysis verified the melatonin-mediated regulation of chi-let-7d-5p and chi-miR-1388-3p in the hair papilla cells of the goat and their pathways,which revealed the molecular regulatory mechanism of melatoninmediated miRNAs in the growth of hair follicles of the goat,and provided new insights into the interaction network of miRNAs and mRNAs in the process of hair follicle development. |
PDF Full Text Request