Identification And Functional Analysis Of LncRNA Related To Development Of Subo Merino's Hair Follicle

Hair follicle(HF)initiation of sheep is depend on a series of complex inter-cellular signaling pathway interaction between epidermis and dermis.Any one ligand,receptor genetic mutations,epigenetic modification and post-translational modification of the protein in signaling pathway will affect sheep hair follicle occurs.Thus,in addition to the current level of interest in the protein-coding mRNA genes,non-coding of long-chain RNA(long non-coding RNA,LncRNA)as a new type of gene expression regulatory factors,it's also to be focused.A lot of research showed that LncRNA plays an important role in organ formation,cell differentiation and disease occurrence;however,the regulation mechanism of LncRNA in the occurrence of Morphology of hair follicle is poorly known.Therefore,in the present study,the hair follicle development and morphological changes of 10 different parts of fetal skin at 65,85,105,and 135 days and lambs skin at 7 and 30 days after birth were studied at the histomorphology level by histological section methods,in order to explore the structure and morphological changes of hair follicles,and to provide a histological basis for the study of hair follicle growth and development and its regulation mechanism.In addition,18 cDNA libraries were sequenced at the transcriptome level by RNA-seq.Screened the sheep differentially expressed LncRNA,predicted the LncRNA target genes,and conducted the functional analysis of target genes by using analysis software and bioinformatics methods.Furthermore,the differential LncRNAs and mRNAs obtained by in-depth sequencing were verified by quantitative real-time PCR,and discussed the mechanism of LncRNA in hair follicle formation of fine wool sheep.Finally,at the cellular level,TCONS₀0202353,TCONS₀453233,TCONS₀0428783,SFRP2,DKK1,and Hoxc13 related to hair follicle development were analyzed.The main results are as follows:(1)Histological study showed that Subo Merino sheep has formed a complete skin structure at the embryonic age of 65 days,primary follicles began to form hair germs;the number of primary follicles increased rapidly at the embryonic age of 85 days,and secondary hair follicles began to develop;the number of secondary follicles increased rapidly at the embryonic age of 105 days,the secondary-derived follicles began to differentiate from the original secondary follicles,and the hair shaft which was emerged through the epidermis can be seen on the pool;most of primary follicles and some of secondary hair follicle had matured at this stage,hair shaft were emerged all parts of the body,and secondary follicles continue to develop and mature at 7?30 days after birth.(2)1,847,973012 raw reads were obtained in 18 cDNA libraries by high-throughput sequencing;after filtering the raw reads by various quality control procedures,a total of 1,726,188919 clean reads were obtained.With the genome alignment,the overall mapping ratio is high or equal to 81%,and after further screening,total of 1,0193 expressed LncRNA were obtained;in which,8653 are the novel LncRNA,while 1540 were the known LncRNA.A total of 471 differentially expressed LncRNAs and 12,812 differentially expressed mRNAs were detected during six different hair follicle developmental phases.The prediction of target genes of differentially expressed LncRNA found that a total of 3,687 potential target genes were identified for 5,852 cis-acting LncRNAs,while 1,722 potential target genes were identified for 4,117 trans-acting IncRNAs.According to the enrichment analysis of differential LncRNA targets,471 differential LncRNA targets are enriched into the 5509 GO term and 268 pathway.Interestingly,we found that 3 pairs of LncRNA-target gene as TCONS₀0202353 and SFRP2,TCONS₀0453233 and DKK1,TCONS₀0428783 and Hoxc 13 may affect the development and morphological changes of hair follicles of Subo Merino sheep.10 differential LncRNAs and 4 mRNAs were validated by qRT-PCR,and the results showed that the expression trend of these LncRNAs and mRNAs were consistent with the RNA-seq results.(3)Functional studies at cellular level showed that over-expression of TCONS₀0202353 significantly decreased the expression of SFRP2 gene;the results of Dual-luciferase reporting system showed that there was a target relationship between TCONS₀0202353 and SFRP2.The results of CCK8,clone formation,and flow cytometry showed that TCONS₀0202353 could promote the proliferation and apoptosis of sheep fibroblasts,which indicated that TCONS₀0202353 could regulate the development of Subo Merino's hair follicle.The histomorphology,LncRNA transcriptome identification and functional analysis of sheep will further promote the study of sheep LncRNA function and gene regulation mechanism.