Modulation Mechanism Of CircCOL1A1-miR-149-5p-CMTM3/AR Axis Regulate The Formation Of Superior-Quality Brush Hair In Yangtze River Delta White Goat

The Yangtze River Delta white goat,also known as the Haimen goat,is the exclusive goat breed in China that can produce superior-quality brush hair.This hair is the finest raw material used for making Chinese calligraphy brushes;this breed has also been praised for this unique characteristic,which has resulted in it receiving a complimentary name:the brush hair goat.Brush hair is usually separated into three categories:Type Ⅰ,inferior-quality hair;Type Ⅱ,normal-quality hair;and Type Ⅲ,superior-quality hair.Superior-quality brush hair,also called Type-Ⅲ hair,is famous for its pure whiteness,brilliant luster and fine elasticity characteristics.Our existing research revealed that 1)androgen secretion and cold stress can stimulate the formation of superior-quality brush hair in Yangtze River Delta white goats,which can further promote and activate the expression of proteins(such asβ-fibrinogen and keratin)and related signaling pathways(such as the Wnt/β-catenin pathway)known to participate in hair growth.2)Dual-specificity phosphatase 6(DUSP6),S100 calcium-binding protein A(S100A),CKLF-like MARVEL transmembrane domaincontaining 3(CMTM3)and heat stress-associated genes(Hsp70)are important for regulating superior-quality brush hair traits.3)CMTM3,also known as CKLFSF3,a high methylation level of CMTM3 could promote the formation of superior-quality brush hair,and also could modulate the transcription level of androgen receptor(AR).4)CMTM3 gene is one of the target genes of miR-149-5p via the database analyzing.MicroRNAs(miRNAs)and circular RNAs(circRNAs),as the important branch of non-coding RNAs family,are of the most popular categories research in non-coding RNAs today.Previous studies have indicated that hair growth is closely associated with hair follicle development,and hair follicle stem cell proliferation,apoptosis and differentiation are critical for hair follicle development.However,the potential regulatory mechanism of ncRNAs(especially in circRNAs and miRNAs)involved in the development of goat hair follicle and the formation of superior-quality brush hair have not been reported.Therefore,based on the results obtained from circRNAs sequencing data and bioinformatic analysis with cervical spine region skin tissues of normal-quality brush hair goats and superior-quality brush hair goats.The miR-149-5p and novel circRNA circCOL1A1 interacting with CMTM3 gene,are selected for further study to explore the role of them in hair follicle stem cells.This study combining with RT-qPCR,Western blotting,Cell cycle and cell apoptosis with flow cytometry assay,Cell immunofluorescence,Immunohistochemistry-paraffin sections,RNA binding protein immunoprecipitation,Nuclear and cytoplasmic RNA extraction,Dual-luciferase assays and technics,to investigative and determine the functions and regulatory mechanism of miR-149-5p and circCOL1A1 in the formation of superior-quality brush hair traits in Yangtze River delta white goat,to construct the regulatory network of "circCOL1A1-miR-149-5p-CMTM3" afterward.The aforementioned results could fill the insufficient of the research in superior-quality brush hair traits via the function and interactive role of non-coding RNAs(miRNAs and circRNAs)and key genes of the traits.The main results of this study are as follows:(1)CMTM3(the key gene involved in the growth and formation of superior-quality brush hair)expression was significantly lower in skin tissues from the cervical spine in superior-quality brush hair goats than that in normal-quality brush hair goats.The role was investigated in hair follicle stem cells by overexpressing or silencing endogenous CMTM3 shows that CMTM3 inhibits proliferation and promotes apoptosis of goat hair follicle stem cells via suppressing the expression of AR and EGF;CMTM3 participates in the regulation of androgen secretion and MAPK signaling pathway by inhibition of AR and EGF to further inhibiting the formation of superior-quality brush hair traits.(2)CMTM3 is the target gene of miR-149-5p,and miR-149-5p expression was obviously higher in skin tissues from the cervical spine in superior-quality brush hair goats than in those from normal-quality brush hair goats.The function and regulatory mechanism were investigated in hair follicle stem cells by overexpressing or silencing miR-149-5p shows that miR-149-5p directly targets the 3’-UTR of goat CMTM3 and upregulates AR expression,miR-149-5p promotes proliferation and inhibits apoptosis of goat hair follicle stem cells via targeting the CMTM3/AR axis;miR-149-5p facilitates β-catenin-induced hair follicle stem cell differentiation by upregulating the differentiation marker genes(β-catenin,C-myc and KRT6)expression.(3)circRNAs sequencing and bioinformatic analysis with skin tissues from the cervical spine in superior-quality brush hair goats and m normal-quality brush hair goats show that 32 differentially expressed circRNAs were selected(13 upregulated and 19 downregulated);GO and KEGG pathway analysis show that the source genes of the differentially expressed circRNAs were mostly enriched in the focal adhesion and platelet activation pathways;besides,the biological processes and morphogenesis of hair follicle development,skin tissues were mainly regulated by the focal adhesion and platelet activation signaling pathway.Functional mechanism analysis shows that:the top 10 upregulated and top 10 downregulated circRNAs-miRNAs sponged interaction networks were harvested by combining with lots of identified miRNAs.Finally,the circRNA-circCOL1A1 was confirmed involved in the superior-quality brush hair trait via Sanger sequencing and functional analysis.(4)The results of circCOL1A1 identification and characterization show that circCOL1A1 originates from the COL1A1 gene,which is located on chromosome 19 in the goat genome,and exons 21,22,23,and 24 generate a head-to-tail closed cyclic structure.Cytoplasmic and nuclear RNA extraction and RT-qPCR assays reveal that circCOL1A1 was mainly located in the cytoplasm of stem cells.RNase-R treatment and actinomycin-D treatment assays show that the expression of circCOL1A1 in hair follicle stem cells was not inhibited and circCOL1A1 was resistant to RNase-R.Functional investigation show that circCOL1A1 suppresses hair follicle stem cell proliferation and differentiation and induces stem cell apoptosis via suppressing the expression of related marker’s genes.(5)The analyzing data from RNAhybrid-v2.1.2 and RegRNA-v2.0 software shows that circCOL1A1 and miR-149-5p shared the only one binding sites,which means circCOL1A1 might function as a sponge to regulate miR-149-5p expression.RNA binding protein immunoprecipitation(RIP)assay,RT-qPCR assay and dual-luciferase assay show that circCOL1A1 acts as a sponge for miR-149-5p in hair follicle stem cells,which then further decreases the expression levels of miR-149-5p.(6)The co-transfected assay with circCOL1A1 and miR-149-5p shows that circCOL1A1 suppresses goat hair follicle stem cell proliferation by sponging miR-149-5p and circCOL1A1 facilitates goat hair follicle stem cell apoptosis by sponging miR-149-5p,which means miR-149-5p could weaken the inhibition role of circCOL1A1 in hair follicle stem cell proliferation and relieve the promotion role of circCOL1A1 in hair follicle stem cell apoptosis.Furthermore,these results indicate that circCOL1A1 controls goat hair follicle stem cell proliferation,apoptosis and differentiation to further inhibit the development of hair follicle and the formation of superior-quality brush hair traits through the circCOL1A1-miR-149-5p-CMTM3/AR axis.