Transcriptomics And Proteomics Analysis Of Goat Mammary Epithelial Cells Induced By Small Molecule Compounds

In this study,goat ear fibroblasts(GEFs)were transformed into mammary epithelial cells(CiMECs)by a small molecule compound-induced transdifferentiation technique.We first characterized the biological properties of mammary epithelial cells obtained from the transdifferentiation of goat fibroblasts.In this paper,the preliminary biological characterization of mammary epithelial cells obtained from the transdifferentiation of goat fibroblasts was carried out,and transcriptomics combined with proteomics analysis techniques were attempted to explore the regulatory mechanism of small molecule compounds inducing the transdifferentiation of goat fibroblasts into mammary epithelial cells,which is expected to lay the foundation for further research on mammary gland developmental differentiation,lactation and mammary bioreactors.1.Transcriptomics analysis of goat mammary epithelial cells(CiMECs)induced by small molecular compoundsThe biological characterization of mammary epithelial cells(CiMECs)obtained from the transdifferentiation of goat fibroblasts were first validated.The results showed that CiMECs could express mammary epithelial cell surfacespecific antigens and secrete milk lipids and milk proteins.Next,we selected cells at important time points in the process of transdifferentiation of CiMECs induced by small molecules(0-8d)to receive samples.Sequencing analysis showed that he genes GLI3,FGF10,FGFR2,MSX2,GATA3,ELF5,etc.that regulate the fate of mammary epithelial cells were significantly up-regulated with the induction time and the related milk protein genes CSN2,CSN3,LTF were finally expressed.Meanwhile,the fibroblast genes FBN1,AXL,VIM,SNAI1,etc.were significantly down-regulated,while the pluripotency-related genes POU5F1,SALL4 and ESRRB were not significant up-regulated.GO analysis of differentially expressed genes showed significant enrichment of GO items such as mammary epithelial cell differentiation and mammary gland alveolus development.,and KEGG analysis showed significant enrichment of mammary epithelial cell development differentiation and lactation-related pathways,including PI3K/AKT signaling pathway,WNT signaling pathway,estrogen signaling pathway,prolactin signaling pathway,etc.2.Proteomics analysis of goat mammary epithelial cells induced by small molecular compoundsIn this study,proteomics analysis technology was used to further study the molecular events associated with the process of small molecule compounds inducing the transdifferentiation of goat fibroblasts into mammary epithelial cells,and combined transcriptomics analysis data to further explore the molecular mechanisms of transdifferentiation.GEFs and CiMECs samples were selected for proteomics sequencing,and differentially expressed proteins(DEPs)analysis showed that marker proteins of luminal cells such as KRT8,KRT18,KRT19,ITGB1,and markers proteins of basal cells such as KRT5,KRT14,and CD44 were up-regulated in CiMECs,while LTF,a protein related to mammary lactation,was also significantly up-regulated.GO functional enrichment showed that DEPs were mainly enriched in glycerolipid biosynthesis process,epithelial cell development,protein transmembrane transport and other GO terms.KEGG Functional enrichment analysis showed that DEPs were mainly enriched in oxidative phosphorylation,steroid biosynthesis and other pathways.Joint analysis of transcriptomics and proteomics showed that differential m RNA/proteins were mainly associated with GO terms such as lipid metabolism,lipid biosynthesis and cell adhesion,and with pathways such as amino acid metabolism and PI3K/AKT signaling pathway.In summary,the present study found that small molecule compounds transdifferentiate fibroblasts into lactating mammary epithelial cells by breaking the fibroblast-specific gene expression program and simultaneously activating the mammary epithelial cell-specific gene expression program.The activation of genes such as GLI3,FGF10,MSX2,GATA3 and signaling pathways such as PI3K/AKT and WNT play a key role in the induction of the transdifferentiation of goat fibroblasts into mammary epithelial cells by small molecular compounds.