CircRNA通过miR-148a/ miR-10a激活BMP7在湖羊毛囊发育中分子机理研究

Hu sheep, a unique sheep breed in China, is well known for its water-wave lambskin pattern. The formation of lambskin pattern was closely associated with the development of hair follicle, but currently its molecular mechanism underlying the development of hair follicle is unknown. Based on our previous studies on the functions of BMP7 in hair follicle development, this project intends to elaborate the effect mechanism of action of miR-148a/ miR-10a in specifically activating BMP7 through various experimental methods including nucleosome shuttle. The functions of candidated circRNA and miR-148a / miR-10a will be confirmed by monitoring the cell proliferation of wool papilla cells,etc. The regulatory effects on the circRNA-miR-148a/ miR-10a-BMP7 will be verified by miRNA pull-down experiments, etc. And the miR-148a and miR-10a will be knockout by CRISPR / Cas9 technology in wool papilla cells and Hu sheep to verify their functions and the relationship among circRNA-miR-148a/miR-10a-BMP7. The results of this study will reveal the role of circRNAs and functions and expression mechanism of BMP7 in the development of Hu sheep lambskin hair follicles , and will provide the theoretical evidence for the excavation, development and protection of lambskin germplasm resources of Hu sheep.

湖羊是我国特有的白色羔皮用绵羊品种，以生产水波状羔皮著称于世。羔皮花纹形成与毛囊发育密切相关，但目前对其分子机制的了解甚少。本项目基于前期对BMP7在毛囊中的功能等研究基础上，拟通过核质穿梭等实验，阐明miR-148a/10a特异性调控BMP7的作用机制；通过检测羊毛乳头细胞的细胞增殖等实验，来明确候选circRNA、miR-148a/10a的功能；利用miRNA pull down等实验，验证circRNA—miR-148a/10a的调控关系；利用CRISPR/Cas9技术分别在羊毛乳头细胞和湖羊体内进行miR-148a 和miR-10a敲除，验证其功能以及circRNA—miR-148a/10a—BMP7之间的调控关系。研究结果将揭示circRNA通过miR-148a/10a作用于BMP7在湖羊羔皮毛囊发育中的功能、表达调控机制，为挖掘、开发和保护湖羊羔皮种质资源提供理论依据。

羔皮性状是湖羊特色性状，受毛囊生长发育调控，毛乳头细胞是毛囊生长发育的核心细胞。本项目在前期对miR-148a/10a和BMP7研究的基础上，构建并验证毛囊生长发育ceRNA网络机制以及其他候选miRNA与基因在毛囊发育中的作用，主要研究结果包括：.（1）在初步验证了miR-148a/10a靶向BMP7的基础上，利用补救实验进一步验证了miR-148a/10a可以结合BMP7抑制毛乳头细胞的增殖。.（2）对湖羊羔羊直毛和小花皮肤毛囊组织进行全转录组测序，筛选出了114个差异表达circRNAs，通过生物信息学分析构建circFTO/circCSPP1-miR-148a/10a-BMP7调控网络，利用双荧光素酶和补救实验证实了circFTO/circCSPP1可以竞争性结合miR-148a/10a调控BMP7表达促进湖羊毛乳头细胞增殖。.（3）利用CRISPR/Cas9技术在细胞和个体水平进一步验证了ceRNA网络能够调控湖羊毛乳头细胞增殖，进而影响毛囊的发育。.（4）在直毛和小花毛囊中利用单细胞转录组测序挖挖掘了74个差异基因，鉴定了19个毛囊细胞群，验证了IGFBP3参与毛乳头细胞体外凝集，PAPPA2调控IGFBP5影响毛乳头细胞增殖。.（5）利用双荧光素酶和补救实验验证了miR-143靶向CUX1调控毛乳头细胞的增殖；揭示了转录因子Sp1、C-MYC和Krox20能够调控CUX1核心启动子区活性影响CUX1表达进而调控湖羊KRT25、KRT71影响毛乳头细胞的增殖。