The Molecular Mechanism Of Transdifferentiation Of Mammary Preadipocytes Into Mammary Epithelial Cells In Dairy Goats By Multiomics

The mammary gland is the only organ that secretes milk in mammals.The mammary gland consists of many different types of cells,including epithelial cells,adipocytes,vascular endothelial cells,fibroblasts,and immune cells.During pregnancy,lactation,and degeneration,with changes in hormones in the body,adipocytes and epithelial cells can undergo transdifferentiation in mammary gland.A substantial number of MECs with lactation function during pregnancy and lactation,30% of which are derive from the ductal stem cells and 70% from the transformation of adipocytes.This shows,the transdifferentiation of mammary adipocytes is one of the important sources.The amount of mammary preadipocyte transdifferentiation into MEC plays an important role in determining milk production.However,the molecular mechanism of the transdifferentiation of breast precursor adipocytes into mammary epithelial cells has not been reported.Therefore,GMECs and preadipocytes were isolated from goat mammary gland and 3D cultured on Matrigel respectively.The culture medium of GMECs was collected as the conditioned medium to induce the transdifferentiation of mammary preadipocytes.The success of transdifferentiation was verified by changes of cell morphology and subcellular organelles,and immunofluorescence signals of the epithelioid marker protein KRT18.Then,mammary preadipocytes pre-and post-transdifferentiation were collected for transcriptome and whole genome DNA methylation sequencing,DEGs and DMGs were screened,and the candidate TF of DEGs and DMGs were predicted using motif enrichment analysis.The proteome in conditioned medium was also detected by protein mass spectrometry.By integrating transcriptome,DNA methylation dataset and secretory proteome,an epigenetic regulatory network was constructed,candidate genes,TF and proteins regulating the transdifferentiation of mammary preadipocytes into MECs were screened.Finally,the function of SPPL3,a potential candidate gene for transdifferentiation,was verified.The following are the main findings of this study:(1)Isolation,identification and induced differentiation of GMEC: the GMECs were isolated from goat mammary gland,and the immunofluorescence were identified by the marker protein KRT18 of the epithelial cells.It was found that the MECs were highly expressed KRT18.GMECs were inoculated in Matrigel for 3D culture and observe under a live cell workstation.At 2th d,GMECs were randomly scattered in Matrigel;at 4th d,GMECs gradually gathered into clusters;at 6th d,GMECs aggregate into larger cell clusters;at 8th d,the cell mass formed an acinar structure and appeared an acinar cavity.The results showed that GMECs could spontaneously induce the formation of acinar structures and acinar cavities.(2)Isolation,identification and induced transdifferentiation of mammary preadipocytes: the preadipocytes were isolated from goat mammary gland,through oil red O staining of lipid droplets,Western blotting and immunofluorescence staining of marker proteins DLK1 and CD34,it was found that mammary preadipocytes had differentiation potential and were highly expressed in DLK1 and CD34.Mammary preadipocytes were inoculated in Matrigel for 3D culture and induced using conditioned medium.At 0th d of observation under a living cell workstation,the cells showed a network emission shape with significant irregular antennae;At 4th d,the tentacles of the cells began to contract and gather into clusters;At 8th d,the cells grew in a swirling pattern,aggregating into multiple cell clusters,and forming acinar structures and acinar cavities similar to those formed by GMECs.The immunofluorescence staining of transdifferentiated mammary preadipocytes also showed strong KRT18 signals,and under TEM,many microvilli,acrosomal granules,desmosomes connecting adjacent cells in the cytoplasm,and a large number of mitochondria were observed.The results showed that mammary preadipocytes were successfully induced to transdifferentiate into MECs and form acinar structures.(3)Transcription and DNA methylation profiles of goat mammary preadipocytes pre-and post-transdifferentiation: the results showed that 887 genes were specifically expressed in Gpredipocyte group,and 822 genes were specifically expressed in Predipocyte group.Compared with the Preadipocyte group,2238 DEGs were upregulated in the Gpreadipocyte group,which were mainly related to immune response,cell cycle,cell adhesion,morphogenesis and organogenesis;2099 DEGs were downregulated,which was closely related to cell cycle and cell adhesion.Through the enrichment analysis of TF binding motifs,it was found that 13 TFs were significantly enriched in upregulated DEGs,and11 TFs were enriched in downregulated DEGs.DNA methylation sequencing was performed on goat mammary preadipocytes pre-and post-transdifferentiation.A total of 577 DMLs and 17 DMRs were screened in Gpredipocyte group,and 152 DMGs were annotated,which were mainly involved in cell cycle,cell adhesion,the process of maintaining cell structure and stability.And 15 TFs were enriched in25 DMGs methylated in the promoter.Through the interaction between TFs and DMGs,and the relationships between 14 TFs and 15 DMGs were determined.(4)Screening of functional genes during transdifferentiation of goat mammary preadipocytes: The correlation between gene expression and DNA methylation level of corresponding genes was obtained by calculating Pearson correlation coefficient.There were32 genes negatively correlated,which were annotated on the establishment of planar polarity,the regulation of cell adhesion,MEC differentiation,mammary epithelial development and mammary alveolar development;37 genes were positively correlated,which were annotated on cell adhesion,intercellular junction,cell migration and cell shape regulation.DEGs and DMGs intersected to 29 genes.Based on the correlation analysis and the intersection of DEGs and DMGs,3 genes were screened to participate in the methylation mediated transdifferentiation of mammary preadipocytes into MECs,SPPL3,RND1 and PLCL1.SPPL3 promoter had 8 DMLs,and8004918 was decreased(P<0.05);The methylation of 30564254 site in RND1 and 9627299 site of PLCL1 was increased(P<0.05).These DMLs may be involved in the epigenetic changes of transdifferentiation.(5)Construction of epigenetic regulation network in transdifferentiation process:The secretory proteins of conditioned medium were detected by protein mass spectrometry,and 32 proteins were annotated.The 4 candidate proteins were obtained,including ACTG1,ALB,NID1 and HSP90AA1,which were related to cell adhesion,establishment of cell polarity and morphogenesis of polarized epithelium.Then,an epigenetic regulatory network was constructed by integrating DNA methylases,DMGs and secretory proteins for analysis.(6)Effect of SPPL3 on transdifferentiation of goat mammary preadipocytes: The overexpression vector pc DNA3.1-SPPL3 was constructed and transfected into mammary preadipocytes,and induced transdifferentiation using conditioned medium,which inhibiting the m RNA expression of KRT18,Plin2,ACTG1 and Klf11(P<0.05).The results showed that SPPL3 inhibited the transdifferentiation of mammary preadipocytes into MECs.In conclusion,GMECs can spontaneously induce the acinar formation and acinar cavity in vitro.In the presence of GMEC medium,mammary preadipocytes can be transdifferentiated into MECs.By integrating the transcriptome,DNA methylation dataset,and the proteome,epigenetic regulatory network was constructed,3 candidate genes and 4candidate proteins were screened.The candidate gene SPPL3 was also verified,and it was found that SPPL3 inhibited the transdifferentiation of mammary preadipocytes into MECs.This study provides new insights into adipocyte plasticity,accumulates data for further revealing the lactation regulatory mechanisms of the mammary glands of dairy goats,and provides important references for revealing the lactation regulatory mechanisms of other mammals.