Molecular Mechanism Of LncRNA2919 Regulates The Cyclic Regeneration Of Hair Follicle In Angora Rabbit

Hair follicles?HFs?are complex,unique and growing cyclically organs during growth and development processes in mammals.Because of the high self-renewal ability and obvious structure characteristics,HF could act as a good scientific model for explaining the problems of life,such as cell proliferation,cell differentiation and cell apoptosis.The wools of rabbit are very popular with consumers,due to the characters,for instance soft,warm,fine,thin,beautiful and chromatophil.However,the demands for wools have been increased with the improvement of people's living standards and development of the wool textile industry.The improvement of production and quality of wools have been become an urgent issue to be addressed.Therefore,the key factors which affecting the growth of rabbit wools should be explored,and it is important to clarify the molecular mechanism of HF growth and development of improving the wool production and competitiveness in rabbit industry.Long non-coding RNAs?lncRNAs?is a non-coding RNA which contains more than 200bp,and it does not have ability to encode proteins.LncRNA could regulate cell proliferation,cell apoptosis,cell differentiation,and cell migration in a variety of cells.At present,lncRNA could participate in ceRNA regulatory network,DNA methylation and HF development related signaling pathways in various animals,such as human,mouse and goat.Moreover,lncRNA regulates the cell cycle,cell proliferation and cell differentiation in dermal papilla cells and HF stem cells.But the mechanism of lncRNA involved in the biological process of HF in rabbit has not "been reported.Therefore,the differentially expressed?DE?ncRNA and mRNA in the different developmental stages during the HF cycles in Angora rabbit were obtained by the whole-transcriptome.The novel lncRNA lncRNA2919 was obtained for the further study.The molecular mechanism of lncRNA2919 regulated the HF cyclic regeneration was revealed from in vitro to in vivo.The epigenetic regulation of DNA methylation in the promoter of lncRNA2919 and the trans transcriptional regulatory network of lncRNA2919-STAT1-KRTAP11-1 which could influence the HF cyclic regeneration has been investigated,in order to fill the insufficient of study in HF development via the level of lncRNA1.The HF cyclic regeneration related factors in Angora rabbit were identified by the whole transcriptome sequencing.In order to obtain the HF development related factors in Angora rabbit,the dorsal skin of Wanxi Angora rabbits were shaved and entered anagen for establishing the HF synchronization model.By the measure of wool length,HE staining of paraffin section and the expression detection of HF cycle specific genes?LEF1?SFRP2?TGF-?1 and KRT16?,the different stages of HF cycle of Angora rabbits,such as anagen?0 days to 110 days?,catagen?120 days to 130 days?and telogen?140 days to 150 days?were determined.The dorsal skins at different stages?days 90,130 and 150?during HF cycle were used for the whole-transcriptome,the DE ncRNAs and mRNAs related to the HF cycle were obtained,which included 111 DE lncRNAs,247 DE circRNAs.97 DE miRNAs and 1168 DE lncRNAs.The expression levels of DE ncRNAs and mRNAs at different hair follicle developmental stages were consistent with the results of whole transcriptome by qRT-PCR.In the GO analysis,DE ncRNAs and mRNAs were enriched in the skin and HF development related GO terms,such as HF development,HF cycle.HF development regulation and skin development.KEGG pathway analysis showed that the DE ncRNAs and mRNAs were enriched in the HF development related signaling pathways,such as Wnt signaling pathway.Hedgehog signaling pathway and MAPK signaling pathway.At the same time,the interacted regulation relationships between ncRNAs and mRNAs were predicted,ceRNA regulatory networks such as lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA were constructed,which could reveal the interacted regulation between DE ncRNAs and mRNAs during HF cycle.In the result.lncRNA2919 could highly expressed in the catagen during HF cycle,which could act as a key factor to play an important role in the HF development2.The identification of lncRNA2919 and its effect on the HF growth in Angora rabbit.Based on the results of whole-transcriptome,lncRNA2919 could act as the candidate lncRNA for the further study.The full-length sequence of lncRNA2919 was 1933bp obtained by RACE technology.lncRNA2919 located at 161144649?161147952 loci on chromosome 14.Bioinformatics prediction and labeling vectors verification experiment showed that lncRNA2919 could not encode proteins.The detection of lncRNA2919 expression level between nucleus and cytoplasmic showed that lncRNA2919 was mainly localized in the nucleus.In rabbit dermal papilla cell?DPC?,lncRNA2919 could downregulate BMP2.CCND1,LEF1 and WNT2 genes?P<0.01?.lncRNA2919 could also inhibit cell proliferation,promote cell apoptosis.The adenovirus of overexpression and knockdown of lncRNA2919 was subcutaneously injected into the dorsal skin of HF synchronization model of Angora rabbits.The results showed that lncRNA2919 could reduce the density and depth of HF,delay the growth of hair shaft,extend the transition from telogen to anagen,inhibit the cyclic regeneration of HF.What's more,lncRNA2919 could significantly upregulate the mRNA expression level of FGF5,KRTAP11-1 and STAT1?P<0.01?,significantly downregulate the mRNA expression level of LEF1 and WNT2?P<0.01?,and downregulate the mRNA expression level of BCL2 and CCND1?P<0.05?,which indicated that lncRNA2919 played a negative role in the cyclic regeneration process of HF in Angora rabbits.3.DNA methylation could prevent EGR1 binds to the region of lncRNA2919 promoter.which influenced transcriptional regulation of lncRNA2919.In order to further reveal the regulatory mechanism of lncRNA2919 promoter during the cyclic regeneration of HF in rabbit.The methylation of lncRNA2919 promoter at different stages of HF cycle in Angora rabbits were detected by Bisulfite sequencing.The results showed that there were two CpG island in lncRNA2919 promoter,the CpG₃ site in CpG island 2 was negatively correlated with the expression of lncRNA2919 in different stages of HF cycle?Pearson Correlation Coefficient was-0.99?.DPC was demethylated by methyltransferase inhibitor 5-Aza-dc,which could significantly inhibit the mRNA expression level of DNMT1?P<0.01?and significantly increased the expression of lncRNA2919 in DPC?P<0.01?,indicating that hypermethylation level of lncRNA2919 in DPC.Furthermore,the results of luciferase reporter showed that the-2069 to-849 loci in the lncRNA2919 promoter was the highest luciferase activity region.Demethylation could increase the luciferase activity in-2069 to-849 loci by 5-Aza-dc treatment,while the CpG₃ site in CpG island 2 located in this region.By the transcription factor?TF?prediction of CpG₃ site in CpG island 2,TF Early growth response 1?EGR1?located in this CpG site.What's more,the results showed that EGR1 could specifically bind the promoter region?-1121?-1108?of lncRNA2919 and significantly promote the activities of lncRNA2919 promoter region?P<0.05?using site mutation,luciferase reporter and EMSA technique.Demethylation and TF EGR1 could significantly upregulate the expression level of lncRNA2919 in DPC?P<0.01?.Thus,the results revealed that DNA methylation regulated the binding relationship between TF EGR1 and lncRNA2919 promoter region,which regulated the transcription of lncRNA2919,and participated in the HF regeneration process in rabbits4.STAT1 was recruited by lncRNA2919 to form a compound,which could regulate transcriptional expression of KRTAP11-1.To further analyze the downstream signaling transduction network of lncRNA2919,binding proteins of lncRNA2919 was screened by RNA pull-down and mass-spectrometric,the results showed that many skin and HF development related proteins,such as Signal transducer and activator of transcription 1?STAT1?and Keratin 16?KRT16?may bind with lncRNA2919.The overexpression of lncRNA2919 significantly increased the mRNA and protein expression level of STAT1?P<0.01?,the combination between STAT1 and lncRNA2919 was verified by RIP assay.According to the function prediction of translcis by whole-transcriptome,the results showed that there was a trans regulation relationship between lncRNA2919 and KRTAP11-1.Moreover,the function prediction showed that lncRNA2919 was located about 160kb in downstream of KRTAP11-1,and lncRNA2919 was positively correlated with KRTAP11-1 at different stages of the HF cycle?Pearson Correlation Coefficient was 0.96?.the overexpression of lncRNA2919 could significantly increase the mRNA expression level of KRTAP11-1?P<0.01?.KRTAP11-1 could significantly upregulate the mRNA expression level of KRT16 and KRT17?P<0.01?,and significantly downregulate the mRNA expression level of LEF1,WNT2,CCND1 and BCL2?P<0.01?.Moreover,KRTAP11-1 could inhibit DPC proliferation and promote DPC apoptosis.Furthermore,the results of luciferase reporter showed that-2490 to-2160 was the core region in KRTAP11-1 promoter,and prediction of TF showed that STAT1 could bind with lncRNA2919 located in the region.The results of site mutation,luciferase reporter and EMSA showed that STAT1 could bind to the KRTAP11-1 promoter region and significantly upregulate the transcriptional activity of KRTAP11-1?P<0.05?.Based on the analysis of interacted protein and regulation of trans,it was proved that lncRNA2919 could recruit STAT1 to form a compound which located in the KRTAP11-1 promoter region,and lncRNA2919 may involve in the cyclic regeneration of HF through the trans regulatory axis of lncRNA2919-STAT1-KRTAP11-1 in Angora rabbits.