Identification Of LncRNAs And Expression Regulation Analysis Of Key Genes In The Development Of Yak Hair Follicle Cycle

The seasonal cycle growth and shedding of yak fluff is of great significance to the adaptability of yak to alpine environment.Similar to the hair follicle(HF)cycle on mammalian,the development of yak HF cycle also mainly includes three stages: anagen,catagen,telogen.As an important barrier against the external environment,yak skin and hair may also form their unique molecular properties under long-term adaptation to the cold environment.In recent years,it has been found that long non-coding RNA(lncRNA),as an important regulatory gene,also plays an important regulatory role in hair follicle development.In order to explore the molecular mechanism of yak HF cycle,the expression profile of lncRNAs in skin tissues at different stages of yak hair cycle was detected in this study,and the potential function of differentially expressed lncRNAs in yak HF cycle was analyzed.Meanwhile,the key genes involved in regulating yak hair follicle cycle development at different stages were further screened by construction of the m RNA-lncRNA co-expression network.Then,dermal Papilla cells(DPCs)and hair matrix cells(HMCs)on yak were isolated and cultured,and the differentially expressed m RNA and miRNA between DPCs and HMCs were detected by RNA-seq,to further exploring the molecular regulation mechanism of yak HF cycle by analyzing the biological function and signals communication of DPCs and HMCs during the development of HF cycle.The main results are as follows:1.The lncRNAs of skin tissues in different stages including five time points of yak HF cycle development were detected.A total of 2884 lncRNAs were identified,and 728 differentially expressed lncRNAs(DELs)were screened using Fold change(FC)>2 and P-value <0.05 as thresholds.Subsequently,DELs between catagen,telogen and anagen were obtained and the GO and KEGG enrichment analysis of their cis-regulated target genes were performed.Enrichment analysis showed that the target genes of DELs were mainly involved in the biological process of keratinization,keratin filaments,thiamine transport and protein ubiquitination.The KEGG pathways including VEGF,Wnt,regulation of pluripotent stem cell signaling pathways were enriched.2.Based on the expression profile of differentially expressed lncRNAs and m RNAs during the yak hair cycle,the weighted gene co-expression network analysis(WGCNA)was used to construct the lncRNA-m RNA co-expression network,and four time-specific gene modules closely associated with differentially stages,respectively,were identified.Hub genes of different modules were screened as key regulatory genes in corresponding periods.Meanwhile,five key transcription factors including WNT5 A,FOXN1,HOXC13,DLX3 and OVOL1,in the co-expression network associated with the anagen(Oct.),were identified to be specifically expressed in skin tissue.The expression locations and abundance of DLX3 and OVOL1 were detected to be changed during hair follicle development.These results indicated that the Hub genes may play a certain regulatory role in yak hair follicle cycle development,and also illustrate the reliability of the screened Hub gene in regulating hair follicle development.3.DPCs and HMCs were isolated from yak skin,and the expression pattern of m RNAs and miRNAs between DPCs and HMCs were detected,4600 differentially expressed m RNA(DEGs)were screened using the threshold of q-value <0.05 and |log2FC|>1,of which 2256 were up-regulated,and2335 down-regulated in DPCs.GO and KEGG enrichment analysis showed that up-regulated genes in DPCs were mainly enriched in GO items of cell adhesion,collagen and extracellular matrix,HMCs up-regulated genes were mainly enriched in the GO items related to intercellular signal transduction,establishment of skin barrier and cytoplasmic membrane.In addition,Wnt,TGF-β,platelet activation,ECM receptor interaction,Tight junction and other KEGG signaling pathways related to HF development were enriched in the DEGs.A total of 439 differentially expressed miRNAs were screened,including 276 known miRNAs and 163 predicted novel miRNAs.Target genes of differentially expressed miRNAs were predicted based on the DEGs between DPCs and HMCs.GO and KEGG enrichment analysis results showed that GO terms including Wnt signaling,cell adhesion,cell cycle and skin development were enriched,KEGG signaling pathways such as Notch,Wnt,Melanogenesis and Basal cell carcinoma were enriched,suggesting that differentially expressed miRNAs may participate in the regulation of hair follicle growth and development through the above biological processes and signaling pathways.In addition,multiple marker genes of DPCs and miRNAs specifically expressed in DPCs or HMCs were also identified in this study.4.DPCs and HMCs were treated with different concentrations of Dihydrotestosterone(DHT)and melatonin,respectively.MTT experiment results showed that 20μM and 40μM DHT were the best concentration to promote the proliferation of DPCs and HMCs,respectively.In addition,500pg/m L melatonin had the most significant promoting effect on the proliferation of DPCs,while different concentrations of melatonin had no significant effect on the proliferation of HMCs,indicating that different hair follicle cells have a different response to DHT and melatonin.In addition,when HMCs were treated with a combination of DHT and melatonin,the proliferative effect of DHT on HMCs was significantly inhibited by melatonin,suggesting that melatonin had an antiandrogen effect.To sum up,this study identified and analyzed the lncRNAs and their biological functions during the development of yak hair cycle,the hub genes in different stages of yak HFs cycle were screened,and the molecular biological basis of different functions of DPCs and HMCs and the differences in their responses to related hormones at the cellular level were dected.This result will provide data support for revealing the molecular mechanism of the development of HF cycle and breeding of yak hair traits.