| The cashmere produced by cashmere goats is well-known both domestically and internationally for its whiteness,softness,fineness,and good luster,and is an important foreign exchange earning livestock product in our country.Cashmere goat hair follicles are mainly divided into primary hair follicles and secondary hair follicles.Cashmere is produced by secondary hair follicles.The morphogenesis and development of secondary hair follicles have important effects on the quantity and quality of cashmere.This process is also regulated by a variety of signaling molecules and their related signaling pathways.Therefore,the main contents of this study included:(1)Based on the whole transcriptome sequencing,the expression profiles of circRNA,miRNA and mRNA in the skin tissue of cashmere goats at different developmental stages(45d,55d,65d and 75d)of embryonic skin hair follicles were obtained;(2)Differentially expressed circRNA,miRNA and mRNA in each comparison group were identified,and circRNA,miRNA and mRNA related to the morphogenesis and development of secondary hair follicles were screened based on the morphogenesis of secondary hair follicles at different stages.(3)Functional enrichment analysis and targeted relationship prediction were used to construct circRNA-miRNA-mRNA regulatory network in the signaling pathways related to secondary hair follicle morphogenesis and development of cashmere goats.(4)To resuscitate and culture cashmere goat fibroblasts cell,and establish key circRNA,miRNA and miRNA target gene interference/overexpression cell lines.By detecting the expression levels of miRNA and its target genes,combined with the research results on the effect on cell cycle proliferation and migration,The functional mechanism of circRNA-miRNA-mRNA regulatory network in the morphogenesis and development of secondary hair follicles in cashmere goats was analyzed.The main research results are as follows:1.The expression profiles of circRNA,miRNA and mRNA in skin tissues of cashmere goats at different embryonic stages(45d,55d,65d and 75d)were established by full transcriptomic sequencing.There were 92(59 up-regulated,33 down-regulated),163(61 up-regulated,102 down-regulated),65(32 up-regulated,33 down-regulated circRNAs),236(67 up-regulated,169 down-regulated),159(96 up-regulated,63 down-regulated)and 118(76 up-regulated,42 down-regulated)differentially expressed circRNAs;there were 327(181 up-regulated,146 down-regulated),117(66 up-regulated,51 down-regulated),137(90 up-regulated),respectively.47 down-regulated miRNAs),19(5 up-regulated,14 down-regulated),77(36 up-regulated,41 down-regulated)and 141(53 up-regulated,88 down-regulated)differentially expressed miRNAs;there were 4802(2464 up-regulated,2338 down-regulated),4409(2204 up-regulated,2205 down-regulated),1108(771 up-regulated,337 down-regulated,4715(2397 up-regulated,2318 down-regulated),5219(2697 up-regulated,2522 down-regulated)and 881(509 up-regulated,372 down-regulated)differentially expressed mRNAs in the 6 comparison groups of 75dvs45d,75dvs55d,75dvs65d,65dvs55d and 55dvs45d,respectively.qRT-PCR results of randomly selected 4 circRNAs,6 miRNAs and 6 mRNAs showed that the full transcriptome data obtained by high-throughput sequencing was reliable.GO and KEGG enrichment results of differentially expressed mRNAs,differentially expressed miRNA target genes and differentially expressed circRNA source genes showed that these genes were mainly involved in the biological processes of hair follicle morphogenesis,hair follicle maturation,and cell development.It was significantly enriched in Wnt,TGF-β,NF-kappa B and PIK3-AKt signaling pathways related to hair follicle morphogenesis and development.2.Based on the differentially expressed circRNA,miRNA and mRNA identified in each comparison group,a total of 118 circRNA,66 miRNA and 664 mRNA that might be related to the morphogenesis and development of secondary hair follicles were screened out according to the morphological changes of primary and secondary hair follicles at different periods.The GO enrichment results of 664 differential genes showed that a total of 2713 GO teams were enriched,and 389 of them were significantly enriched.Biological processes mainly include stem cell differentiation,positive regulation of cell proliferation,positive regulation of NF-kappa B pathway,etc.Cell components include cytoplasmic matrix,etc.Molecular functions include ATP binding,DNA binding and protein binding.KEGG pathway analysis showed that 63 KEGG pathways were co-enriched,and 14 of them were significantly enriched,including Notch and TGF-βsignaling pathways related to hair follicle development.3.Based on the screening of circRNA,miRNA and mRNA and important signaling pathways related to secondary hair follicle morphogenesis and development,a total of 503 circRNA-miRNA-mRNA regulatory networks were constructed by bioinformatics,which were composed of 13 circRNA,21 miRNA and 111 mRNA in the(Notch,Wnt,TGF-β and NF-kappa B)signaling pathways related to secondary hair follicle morphogenesis and development in cashmere goats.The regulatory network related to key factors of Notch signaling pathway DLL4 and Notch2 gene was selected for verification.qRT-PCR results showed that the relative expression correlation coefficients of circRNA888-chi-miR-27b-3p,circRNA2034-chi-miR-27b-3p,circRNA5712-chi-miR-27b-3p,circRNA2109-chi-miR-30e-5p,chi-miR-27b-3p-DLL4,chi-miR-30e-5p-DLL4,chi-miR-145-5p-DLL4,chi-miR-103-3 p-Notch2 and chi-miR-10b-5p-Notch2 were-0.80,-0.72,-0.51,-0.57,-0.87,-0.88,-0.54,-0.50 and-0.57,respectively.The results preliminary showed that all groups had a targeting relationship.4.Based on the construction and preliminary verification of the ceRNA regulatory network of circRNA related to the morphogenesis and development of secondary hair follicles in cashmere goats,the functional role of miRNA in the regulatory network was first explored.Dual-luciferase reporter gene results showed that chi-miR-30e-5p and DLL4,chi-miR-27b-3p and DLL4 had targeted binding relationships.However,chi-miR-145-5p and DLL4,chi-miR-10b-5p and Notch2,chi-miR-103-3p and Notch2 had no targeted binding relationship.In fibroblast cell lines,chi-miR-30e-5p and chi-miR-27b-3p had good overexpression and interference effects,and had significant negative regulatory effects on their target gene DLL4.It was further confirmed that chi-miR-30e-5p and chi-miR-27b-3p may participate in the morphogenesis and development of secondary hair follicles in cashmere goats by targeting DLL4.5.Based on the above functional verification results of related miRNA in ceRNA regulatory networks,the source composition analysis showed that circRNA5712,circRNA2109,circRNA888 and circRNA2034 were exon circRNA and were real.Dual-luciferase reporter gene results showed that circRNA888,circRN5712 and circRNA2034 all had a targeting relationship with chi-miR-27b-3p,while circRNA2109 had no targeting relationship with chi-miR-30e-5p.Four optimal interference stable transmutation fibroblasts cell lines(circRNA5 712-sh 1,circRNA8 8 8-sh3,circRNA2034-sh1 and DLL4-sh1)were established.Through qRT-PCR detection,DNA staining,CCK8 method,and cell scratch test verification,it was found that circRNA888,circRN5712,and circRNA2034 may regulate the morphogenesis and development of secondary hair follicles in cashmere goats through the chi-miR-27b-3p-DLL4 Notch signaling axis. |
PDF Full Text Request