Inner Mongolian Cashmere Goat Villus Growth Cycle And Growth Initiation Regulatory Research Based On MicroRNA-mRNA Co-analysis

The differentially expressed genes in Cashmere goat skin has correlation with follicle growth cycle and the villus quality.Many factors have to regulate the gene expression,like microRNA has regulate the gene expression in the post-transcriptional level.micro RNA and mRNA has construct the complicated regulatory network in the skin to regulate the hair follicle growth cycle and development.Use High-throughput sequencing technology to research the cashmere goat skin gene expressed pattern,and construct the microRNA-mRNA regulated network to select the gene which correlate with the follicle growth cycle and villus quality,open an new ideas to find the regulatory gene.This research adopted the High-throughput sequencing method analysis three female Arbas cashmere goats twelve months skin samples and annotation the transcripts,analysis the differentially expressed gene in twelve months skin sample.And Co-analysis the data from telogen and anagen period skin’s RNA-Seq and microRNA-Seq,enrichment analysis the differentially expressed micro RNA’s target genes and construct the micro RNA-mRNA regulatory network.From the regulatory network to select the gene and micro RNA which are correlated with the villus quality and follicle growth cycle,then verify it.The main results as following:1.The skin RNA-Seq output 230 million high quality reads,obtained 30.7GB total data,each sample obtained average 2.6GB data.Through by the de novo assemble obtained 55541 transcripts,105854 isogenes,transcript average length is 1956.15 bp,each sample more than 82% clean reads map to the transcripts.2.The transcripts GO annotation show that the gene expressed in skin’s biological process mainly is metabolic process,biological regulation and response to stimulus.Cellular component mainly is cell and membrane.Molecular function mainly is binding and catalytic activity.The transcripts COG annotation show that the skin homologous proteins function mainly is transcription,posttranslational modification,general function and signal transduction mechanisms.The transcripts KEGG pathway analysis show that the Wnt,MAPK,Notch,TGFβsignal pathway has enrichment most transcripts.3.Analysis the each sample’s transcripts expression show that between the skin samples has differentially expressed genes,there are four times great DEGs expre ssion issue,on the period of follicle start to growing has great DEGs expression issue,and on the catagen and telogen period the DEGs expression issue has smoothly comparatively.Interested is the March skin sample has most DEGs which are keratin protein genes and zinc finger protein genes.The keratin associated protein gene’s expression pattern has correlation with follicle growth cycle.4.Co-analysis the telogen and anagen skin RNA-Seq data with microRNA-Seq data got the 12927 genes which are targeted by differentially expressed microRNA,among this there are 6951 DEGs and 5114 DEGs which has negative relationship with their targeted microRNA expression.The interaction network between DEGs and micro RNA are complicated,there are both negative and positive expression pattern relationship between DEGs and microRNA.Mean the follicle star to growth has been regulated by large amount of genes,between the genes has balance effection,the threshold of the gene expression are important for follicle growth.5.From the differentially expressed microRNAs’ target gene annotation data,there are more than two-thirds m RNA has regulatory relationship with micro RNA.The differentially expressed micro RNA’s target gene KEGG pathway enrichment analysis show that insulin signal pathway has most enrichment value,And there are 187 DEGs are annotate in the Wnt signal pathway,this enrichment value are higher.The oxidation-reduction process,DNA binding and catalytic activity,integral component of membrane and nucleus annotations has own large proportion differentially expressed microRNA’ negative control genes.6.Construct the differentially expressed microRNA’s negative control target gene GO annotation interaction network and KEGG pathway interaction network and Wnt gene interaction network.Select out the CHP1,SIAH1,FZD6 and SMAD2 which are annotate in the Wnt signal pathway and up regulated,and located on the interaction center in the Wnt gene interaction network.And the four gene are both negative regulated by miR-195.7.Analysis the villus main ingredient KRTAPs expression data with the microRNA data,select out the KRTAP24-1,KRTAP3-1,KANSL1 and EGFR which are negative targeted by miR-195.8.Use RT-PCR to testify the miR-195 expression pattern,the result consistent with microRNA-Seq data,the mi R-195 has down regulate significant(p≤0.01).9.Use RT-PCR method to testify the selected DEGs CHP1、SIAH1、FZD6 and SMAD2 expression pattern,the result consistent with RNA-Seq data.Use RT-PCR method to testify different types of keratin protein genes and keratin associated protein genes expression pattern,find the KHA5、KHA6 and KHB4 are regulate the follicle star to growth,and find the KRT and KRTAP has differentially expression pattern in the different period of follicle growth and the expression pattern has correlation with the follicle cycle.This research first co-analysis the mRNA and microRNA sequencing data to construct the regulatory network and use this method to study the regulatory mechanism of the follicle growth cycle,obtained the CHP1、SIAH1、FZD6 and SMAD2 which are correlate with the follicle growth and the miR-195 which are regulate the follicle growth,make some theory foundation for transgenic breeding and artificial regulation.