Regulation Of Rho Family GTPase 1 In The Transdifferentiation Of Mammary Precursor Adipocytes In Dairy Goat

The amount of lactation by the mammary gland depends on the number and quality of lactating cells.The transdifferentiation of mammary precursor adipocytes into mammary epithelial cells occurs in the mammary tissue during pregnancy and lactation,and the amount of mammary adipocyte transdifferentiation into mammary epithelial cells is decisive for the amount of lactation in the mammary gland.It is important to study the transdifferentiation of mammary adipocytes during pregnancy and lactation in dairy goats,as well as the gene regulatory mechanisms affecting this process,to improve milk production in dairy herds.Our laboratory previously successfully induced transdifferentiation of mammary precursor adipocytes cultured in vitro into mammary epithelial cells and screened for the key gene regulating the transdifferentiation process,RND1(Rho family GTPase 1),and the transcription factor CTCF,by analyzing transcriptome differences in mammary precursor adipocytes before and after transdifferentiation.At present,there is no report on the effect of RND1 on transdifferentiation of mammary precursor adipocytes into mammary epithelial cells in dairy goats.Therefore,this study aimed to investigate the role of RND1 in the process of inducing transdifferentiation of mammary precursor adipocytes into mammary epithelial cells in dairy goats,and to provide a basic information for related studies on the regulatory mechanism of mammary gland development from pregnancy to lactation stage in dairy goats.In this study,primary mammary precursor adipocytes and primary mammary epithelial cells from dairy goat were first used to induce transdifferentiation of mammary precursor adipocytes into epithelial cells using conditioned medium,and then the expression of RND1 in mammary precursor adipocytes before and after transdifferentiation was examined,gene overexpression,Western blot of cells were used to explore the role of RND1 in the transdifferentiation of mammary precursor fat into epithelial cells.The main findings of this study are as follows:1.Mammary precursor adipocytes were induced to transdifferentiate into mammary epithelial cells by preparing conditioned media using media supernatant from cultured mammary epithelial cells mixed 1:1 with DMEM / F12.At day 10 of the induction of transdifferentiation,the morphology of mammary precursor adipocytes changed from elongated spindle to nearly round shape as observed by inverted fluorescence microscopy,accompanied by downregulation of the m RNA and protein expression of the precursor adipocyte marker genes CD34 and DLK1(P < 0.01),upregulation of the m RNA and protein expression of the epithelial cell marker CDH1(P < 0.05)and of the m RNA and protein expression of krt18(P < 0.01).2.Cloning resulted the dairy goat RND1 CDS region,which is 699 bp in length and encodes 232 amino acids.The dairy goat RND1 protein is evolutionarily the least genetically distant from goat,sheep,cattle,and fawn.Examination of the m RNA expression of RND1 in mammary tissue at different lactation periods revealed that RND1 was most highly expressed in the early stage of lactation in dairy goats.3.The eukaryotic pc DNA3.1-RND1 expression vector was constructed,overexpression of RND1 in mammary precursor adipocytes upregulated the m RNA expression of the epithelial marker genes CDH1 and ELF5(P < 0.01)and the epithelial polarity related gene SCRIB(P < 0.05),and downregulated the m RNA expression of the lipid droplet generation related gene FABP4(P < 0.05).Overexpression of RND1 in mammary precursor adipocytes simultaneously induced transdifferentiation,at day 10 of transdifferentiation,overexpression of RND1 promotes the transdifferentiation process,downregulated m RNA expression of FABP4(P < 0.01),and upregulated m RNA expression of CD34,DLK1,as well as genes including PPARG and CEBPA(P < 0.01).4.The full-length sequence 2688 bp of RND1 promoter was cloned,and the core region of the RND1 promoter was found to be located from-329 to-109 bp by truncation analysis using the 5 ’-end flanking sequences in dairy goat.Overexpression of CTCF down regulated RND1 promoter activity(P < 0.01),demonstration that CTCF negatively regulates RND1 expression.In summary,RND1 positively regulates CDH1 and epithelial polarity related gene SCRIB expression in mammary precursor adipocytes,RND1 promotes cell morphology transition to epithelioid cells during the induction of transdifferentiation of mammary precursor adipocytes and inhibits the expression of lipid droplet generation related gene FABP4,while Rnd1 is negatively regulated by transcription factor CTCF.