Screening Of MiRNAs And Identification Of Candidate Target Genes In Skin And Hair Follicle From Liaoning Cashmere Goat And Qianhua Mutton Merino

Liaoning cashmere goat(Capra hircus)is a unique genetic cashmere-mutton breeding with long and fine cashmere,10 cm and 15 ?m.Qianhua mutton merino is a new variety of mutton-fine wool sheep(ovis aries)from Jilin province,with good mutton characteristics and wool fineness,20.47-21.43 ?m.mi RNA is a kind of single strand non-coding small RNA(19-25 nt),which is an essential regulator of cell differentiation,proliferation,apoptosis and cell cycle conversion process,and so on.Hair follicle is an attached and renewable organ of skin with self-renewal and cycling,which controls growth of hair.In order to definite regulation of miRNA on hair follicle development and explore the possible mechanism,screening small RNA was done by High-throughout sequencing,and small RNA libraries were constructed.Differential expression mi RNAs were isolated from different stage skin and hair follicles of Liaoning cashmere goat and Qianhua mutton merino.In this study,target genes of mi R-1298-5p and miR-1-3p were predicted by targetscan v7.0,RNA22 v2.0 and MiRanda(2010)on line predicting software.According to previous documents,five genes(TGFBR1,TGFBR2,FGF2,IGF1 R and FGF14)had been defined to be the candidate target genes.Effect of miRNA and its candidate target genes on skin and hair follicle cycling development were uncovered by Fluorescence quantitative PCR from nucleotide level,and Effect of candidate target genes on skin and hair follicle cycling development were uncovered by western blot.Dual luciferase reporter system experiment was done so as to uncover the regulation between miRNA and its candidate target genes by the change of dual luciferase activity,and further explore the possible mechanism of miRNA and hair follicle development,and provide evidence for regulating cycling development of hair follicle.The main results are as follow:1.Small RNA libraries of hair follicle(anagen,catagen and telogen)were constructed,and 8596054,11039308 and 10478621 clean reads were obtained from Liaoning cashmere goat,10279515?11712275 and 10926258 clean reads from Qianhua mutton merino.The length distribution of small RNA is similar each other.The highest percentage of these small RNAs was 22 nt,the second were 21 nt and 23 nt.2.1616 mature miRNAs were obtained,1397 of them were conservative,219 of them were novel miRNAs.Our findings enriched capra hircus and ovis aries miRNA database.Specific miRNA of anagen,catagen and telogen was respectively 21,23 and 26 from Liaoning cashmere goat,and 15,30 and 27 from Qianhua mutton merino.3.Relative expression of ten miRNAs were determined by fluorescence quantitative PCR.The results of relative expression of ten mi RNAs were basically similar to the results of high-thoughout sequencing,which showed that the results of high-thoughout sequencing were accurate.4.Differential expression of mi RNAs were analyzed by IDEG6 software.TGFBR1,TGFBR2 and FGF2 were on as candidate targets of miR-1298-5p,IGF1 R and FGF14 as candidate targets of miR-1-3p in terms of softwares and documents(Wnt,TGF-?,Notch,SHH,MAPK,BMP,Jak-STAT and TNF).5.The results of fluorescence quantitative PCR showed that relative expression of miR-1298-5p was low expression at anagen,high expression at catagen;miR-1-3p was low expression at anagen,high expression at telogen in Liaoning cashmere goat.miR-1298-5p was low expression at telogen,high expression at catagen;miR-1-3p was low expression at anagen,high expression at telogen in Qianhua mutton merino.TGFBR1,TGFBR2 and FGF2 mRNA were high expression at anagen,low expression at catagen in Liaoning cashmere goat;TGFBR1,TGFBR2 and FGF2 mRNA were low expression at catagen,TGFBR1 and FGF2 were high expression at telogen,TGFBR2 were high expression at anagen in Qianhua mutton merino.Relative expression of IGF1 R mRNA was high expression at telogen,low expression at catagen in Liaoning cashmere goat.Relative expression of FGF14 mRNA was high expression at anagen,low expression at telogen in Liaoning cashmere goat.Relative expression of IGF1 R and FGF14 mRNA were high expression at anagen,low expression at catagen in Qianhua mutton merino.6.The results of western blot showed that relative expression of TGFBR1,TGFBR2 and FGF2 protein were high expression at anagen,low expression at catagen in Liaoning cashemere goat.TGFBR1,TGFBR2 and FGF2 protein were low expression at catagen,TGFBR1 and FGF2 were high expression at telogen,TGFBR2 were high expression at anagen in Qianhua mutton merino.Relative expression of IGF1 R protein was high expression at anagen,low expression at catagen in Liaoning cashmere goat.Relative expression of FGF14 protein was high expression at anagen,low expression at telogen in Liaoning cashmere goat.Relative expression of IGF1 R and FGF14 protein were high expression at anagen,low expression at catagen in Qianhua mutton merino.7.The results of dual luciferase reporter system experiment showed that miR-1298-5p inhibited wild type TGFBR1 and FGF2,miR-1-3p inhibited wild type IGF1 R and FGF14.After mutation of target sites,only dual luciferase activity of FGF2 and FGF14 could recover,which indicated FGF2 was target gene of mi R-1298-5p,FGF14 was target gene of miR-1-3p.Relative expression pattern of miR-1298-5p and mi R-1-3p in Liaoning cashmere goat and Qianhua mutton merino were defined by High-throughput sequencing and Fluorescence quantitative PCR.Relative expression pattern of TGFBR1,TGFBR2,FGF2,IGF1 R and FGF14 nucleotide and protein level were defined by Fluorescence quantitative PCR and Western blot.Target gene FGF2 of miR-1298-5p and FGF14 of miR-1-3p were identified by Dual luciferase reporter system experiment.The results reveal that miR-1298-5p and miR-1-3p may respectivelly affect relative expression of target gene mRNA and protein through binding target sites,resulting in cycling development of hair follicle.