Differential Expression Of MiRNA-125b Between The Skin Of Cashmere Goats And Fine-wool Sheep, Screen And Identify Its Targets

With the development of fur consumer market in the world, the chinese woolhas a good reputation in the international market which is characterized by itsdelicate softness of wool, color white, warm and comfortable, and so on. Amongthe many varieties of wool, hircine and ovine in different textures are welcomed byconsumers. The hair growth process of fine wool sheep and cashmere goats iscomplex and regulated by nutrition, environment, metabolism and gene regulationand many other factors. In this process, the gene regulation has played a decisiverole. In recent years, researchers have done a lot of work in terms of regulationmechanism of hair follicle development, and also has made appropriateachievements. But these developments are mainly concentrated in the traditionalregulatory level while ignoring the study of post-transcriptional regulation. Withthe emergence of miRNA, new regulatory level, namely post-transcriptionalregulation has become the hot spot of research.MiRNA is a new class of endogenous small non-coding RNA which cannegatively regulate eukaryotic gene expression post-transcriptionally via pairing to3’-untranslated region (UTR) of target mRNAs. Then these miRNAs canparticipate in the regualtion of gene expression by inhibiting target gene translationor causing degradation and they play an important role in different biologicalprocesses, such as development, proliferation, differentiation, apoptosis and others.Since the first miRNA, lin-4, was discovered in C.elegans, more and more miRNAin multiple species have successfully been identified. However, the research ofmiRNA on sheep and goats was a little, let alone the research of miRNA in the skintissues of fine-wool sheep and cashmere goats. Nowerdays, the rise ofsecond-generation high-throughput sequencing technology provides a convenientfor identification of these miRNAs so that the study area of miRNA is no longerlimited. Therefore, in this study, based on the previous results of Solexa high-throughputsequencing technology, we randomly selected10differential expression miRNAswhich includes Let-7a-1, miR-184, miR-101a, miR-200a, miR-145, miR-17,miR-451, miR-30b, miR-29b, and miR-125b. Then the relative expression results often miRNAs respectively between fine-wool sheep and cashmere goats wereobtained by fluorescence quantitative PCR in order to further verify the result ofSolexa sequencing. At the same time, differentially expressed significantly(**p<0.01) of miR-125b was chosen as the key research object and it also will besystematically analyzed from in vitro and in vivo. Using the softwares of target geneprediction, such as miRGen V3.0, TargetScan, RNA22and so on, we can predict thetarget genes of miR-125b and then screened these targets which relates to hairfollicle development by GO and KEGG pathway softwares. After that, candidatetarget genes (MXD4、 FGFR2) were selectively identified and verfied byfluorescence quantitative, western-blot and dual luciferase assay system, and amongthem, also using flow cytometry and fluorescence microscopy to detect transfectionefficiency. At last, we also analyzed the sequence of melanocortin4receptor gene(MC4R) in cashmere goats which is a member of the melanocortin receptor family.In order to providing new ideas and research platform of improving the yield andquality of hircine and ovine, in this study we will research the growth anddevelopment of hair follicles in the fine-wool sheep and cashmere goats skin tissuesfrom a new regulation level of miRNA and clarify the mechanism of the differentialexpression miR-125b and its targets in two species. The results showed that:(1) Let-7a-1, miR-184, miR-101a, miR-200a, miR-145, miR-17, miR-451,miR-30b, miR-29b, and miR-125b are both expression in two samples and theexpression levels of ten miRNA in fine-wool sheep are both higher than that incashmere goats. By comparing with the results of Solexa sequencing, we found theexpression trend of ten miRNAs was consistent with the results of qRT-PCR, besideslet-7a-1and miR-184.(2) There were72of miR-125b target genes predicted by the bioinformaticsmethods, and among them, six targets (CBFB, MXD4, FGFR2, EDN1, DLL4,MCRs) were screened by Go and KEGG pathway softwares which were associatedwith hair follicle development. Two of them (MXD4, FGFR2) were chosen to identify. Homology analysis of nucleotide sequence of MXD4and FGFR2usingNCBI BLAST software, the result showed that: Compared with ovis aries, bosgrunniens and so on, the homology were both higher than85%.(3) The results of fluorescence quantitative showed that: At the level of mRNA,targets of MXD4and FGFR2expression in skin tissue of cashmere goats werehigher than that in skin tissue of fine-wool sheep. After that, The results ofwestern-blot showed that: at the level of protein, the expression trend of two targetswas consistent with the results of qRT-PCR.(4) When detected the transfection efficiency of HEK-293T cell line by flowcytometry and fluorescence microscopy, we found that, only the cell proliferate to90%confluence, and final concentration are50nM and200ng of mimics (miRNAsimulacrum) and plasmid vector respectively, the optimal transfection efficiencywould be reached.(5) The results of dual luciferase assay system showed that: FGFR2and MXD4can be identified as the target genes of miR-125b.(6) The results of cloning showed that: the length of MC4R is924bp withoutintron, encoding304amino acids. And there were7transmembrane domains.Homology of nucleotide sequence and deduced amino acid were analyzed, the resultshowed that the homology were both higher than85%with ovis aries, bos gruniens,lupus familiaris, sus scrofa, homo sapiens and so on, especially with ovis aries, thehomology was up to98%which consistent with the result of phylogenetic treeanalysis. Compared with other mammals, there were nine amino acids which existedhigher frequency of mutation. They were located in93,152,160,162,165,182,213,221,230. Among them, there are four mutations (152,160,162,165) located in the secondintracellular loop topology district and three mutations (213,221,230) located in thethird intracellular loop topology. In addition, One mutation (93) located in thesecond transmembrane domain, and the other (182) located in the fourth membranedomain.