Molecular Mechanism Of Transcription Factor HOXC13 In Regulating Periodic Development Of Hair Follicles In Rabbits

China is a large country of rabbit breeding and rabbit hair exporting.Domestic consumers’ demand for high-quality rabbit hair is increasing,and the hair production is closely related to the development of hair follicles.As we known,hair follicles are micro-organs that undergo periodic changes in mammals and are regulated by many factors,affecting hair production and growth.Homeobox C13（HOXC13）gene is reported to regulate the growth and differentiation of hair follicles and hair follicle-associated cells,affecting the morphology and periodic development of hair follicles.Previous studies have found that HOXC13 is involved in the periodic development of hair follicles in rabbits,but the specific regulatory mechanism is still unclear.Therefore,this study analyzed the function of HOXC13 in hair follicle development,verified the regulation of upstream factor miR-129-5p on it,and screened the downstream target genes of HOXC13 protein by ChIP-seq.The molecular regulatory network of miR-129-5p/HOXC13/PRKACB was constructed,which laid the foundation for revealing the mechanism of HOXC13 in hair follicle development in rabbits.The main research results are as follows:1.In order to explore the role of HOXC13 in hair follicle development,qPCR was used to detect the expression of HOXC13 in different tissues（Heart,liver,lung,skin stomach,brain and jejunum）and hair follicle growth cycle（30d、60d、90d、120d、150d and 180d）of Wan-line rabbits.The results showed that the expression of HOXC13 in skin tissues was significantly higher than that in other tissues（P<0.01）,and the expression of HOXC13 was the highest in anagen and the lowest in telogen（P<0.01）.The pcDNA3.1-HOXC13 overexpression plasmid was constructed and transfected into DPCs,the results showed that overexpression of HOXC13 significantly promoted the mRNA expression of hair follicle development-related genes BCL2,WNT2,CCND1 and LEF1（P<0.01）,and inhibited the expression of SFRP2 and TGF-β1（P<0.01）.siRNA interference with the expression of HOXC13 significantly inhibited the mRNA expression of BCL2,WNT2,CCND1 and LEF1（P<0.01）,and promoted the expression of SFRP2 and TGF-β1（P<0.01）.Furthermore,CCK8 and flow cytometry techniques were used to find that HOXC13 promoted the proliferation and inhibited apoptosis of DPCs（P<0.01）,while interference with HOXC13 inhibited the proliferation and promoted apoptosis of DPCs（P<0.01）.Suggested that HOXC13 is involved in hair follicle development.2.The upstream regulatory factors of HOXC13 were further analyzed,and miR-129-5p was predicted to target the 3’UTR of HOXC13 by RNAhybrid online software.In order to confirm whether miR-129-5p is involved in the development of hair follicles in rabbits,qPCR was used to detect the expression of miR-129-5p in different tissues and hair follicles development cycles.The results showed that the expression of miR-129-5p was relatively high in brain and skin（P<0.01）,and the expression level was the highest in catagen,and it was significantly negatively correlated with HOXC13 in the periodic development of hair follicles（Pearson’s R=-0.313,P<0.05）.Overexpression of miR-129-5p significantly inhibited the expression of hair follicle development-related genes BCL2,WNT2,CCND1 and LEF1（P<0.01）,and promoted the expression of SFRP2 and TGF-β1（P<0.01）.Inhibition of miR-129-5p significantly promoted the expression of BCL2,WNT2,CCND1 and LEF1（P<0.01）,and inhibited the expression of TGF-β1（P<0.01）and SFRP2（P<0.05）.overexpression of miR-129-5p inhibited the proliferation and promoted apoptosis of DPCs（P<0.01）.Knockdown of miR-129-5p promoted the proliferation of DPCs and inhibited their apoptosis（P<0.01）.The dual luciferase reporter gene system was further used to verify that miR-129-5p directly targeted HOXC13-3’UTR.qPCR and Western blotting were used to confirm that miR-129-5p inhibited the expression of HOXC13 mRNA and protein in DPCs.These results showed that miR-129-5p could inhibit the expression of HOXC13 mRNA and protein by targeting HOXC13-3’UTR.3.ChIP-seq was further used to screen the downstream target genes of HOXC13 protein.First,the HOXC13-Flag tag vector was constructed and transferred into the DPCs for ChIP-seq detection.The results showed that a total of 9670 enrichment peaks were obtained from HOXC13-Flag peak,and the peak sequence was annotated to the rabbit genome,of which 6.1%was annotated in the promoter region.Association analysis was performed on the genes annotated in the promoter region,and five genes were selected for qPCR verification.The results showed that HOXC13 had regulatory effects on CRH,ENPP1,LDHB,PRKACB and MAP3K2,and PRKACB was selected as the downstream target gene of HOXC13.4.The dual luciferase reporter gene system showed that HOXC13 might target the promoter region+1107-+1596 of PRKACB,and inhibit its transcription.Similarly,functional verification of PRKACB showed that overexpression of PRKACB affected the expression of hair follicle development related genes,which significantly inhibited the proliferation of DPCs and promoted their apoptosis.In addition,co-transfection of miR-129-5p and PRKACB significantly promoted the ability of PRKACB to inhibit the apoptosis of DPCs（P<0.01）.These results show that HOXC13 can target PRKACB to inhibit the expression of PRKACB,promoting the proliferation of DPCs,thereby regulating the growth and development of DPCs,and ultimately participating in the periodic development of hair follicles.This study confirmed that HOXC13 could promote the periodic development of hair follicles,and verified the regulatory effect of upstream factor miR-129-5p on it.ChIP-seq was used to screen the downstream target genes of HOXC13 protein,and PRKACB was selected for functional verification.The molecular regulatory network of miR-129-5p/HOXC13/PRKACB was clarified,which laid the foundation for revealing the mechanism of HOXC13 in the development of hair follicles in rabbits.