Lactation-related Mirnas Screening And Regulatory Network Establishment In Goat Induced Mammary Epithelial Cells

MicroRNAs(miRNAs)are a kind of non-coding RNAs which with a length of about 22 bp.Many studies have shown that miRNA plays an important regulatory role in all process of mammary development and lactation.Goat mammary gland has high milk production and strong post-translational modification ability,which is a good model for in vitro lactation research.To explore the regulation of miRNA on the lactation ability of goat mammary epithelial cells is of great significance for improving the lactation ability of goat individual mammary gland.The previous research results of our group showed that small molecule compounds can transdifferentiate goat fibroblasts into mammary epithelial cells which have lactating function.In this study,the identity of mammary epithelial cells after transdifferentiation was confirmed by miRNA sequencing analysis of cells before and after transdifferentiation.Meanwhile,by further combined analysis of differential mRNA and differential miRNA in the sequencing data,the genes and miRNAs which related to lactation of the transdifferentiated mammary epithelial cells were screened,and the miRNA-mRNA regulatory network that regulates the lactation of the transdifferentiated mammary epithelial cells was preliminarily established.The results of this study lay a foundation for the construction of research platform which to explore the relationship between miRNAs and transdifferentiation and lactation,and are expected to provide some theoretical support for improving the lactation ability of goats.1.MiRNA sequencing analysis of goat mammary epithelial cells induced by small molecule compoundsIn this study,the biological characteristics of cells before and after induction of forskolin,TTNPB,VPA,Repsox and Tranylcypromine were identified.The results of immunofluorescence staining showed that the cells before induction expressed the fibroblast marker antigen Vimentin(VIM),but did not express the mammary epithelial cell marker antigen Epithelial cell adhesion molecule(EPCAM).But after induction,the cells expressed mammary epithelial cell marker antigen EPCAM,but did not express fibroblast marker antigen VIM.The results of saturated Oil Red O staining showed that the cells before induction were not stained red,while the cytoplasm of cells after induction was mostly red.The results of real time quantitative PCR(RT-qPCR)showed that compared with the cells before induction,fibroblast marker genes FBN1 and VIM were significantly down-regulated,and mammary epithelial cell marker genes KRT19,CDH1 and EPCAM were significantly up-regulated in the cells after induction.To further elucidate the changes in cellular miRNA expression levels and cell identity before and after induction,miRNA sequencing was performed on cells before and after induction.The sequencing data were further analyzed for differential expression,differential miRNA target gene prediction,GO functional enrichment analysis and KEGG pathway enrichment analysis.The results showed that compared with those before transdifferentiation,there were135 known miRNAs with significant differences(P<0.05)and 11 new miRNAs with significant differences(P<0.05)in the cells after transdifferentiation.And the target genes of differential miRNAs are mainly enriched in functional items such as developmental process,metabolic process,signal transduction and catalytic activity which related to mammary gland development and lactation,and prolactin signaling pathway,estrogen signaling pathway and PI3K-AKt signaling pathway,which have important regulatory effects on mammary gland development,differentiation and lactation.It indicated that the transdifferentiated cells had changes in miRNA expression level,cell function and cell properties,and possessed the identity of mammary epithelial cells.2.Screening of lactation-related miRNAs and construction of regulatory networksIn this study,based on the phenomenon that small molecular compounds induced goat fibroblasts transdifferentiate into mammary epithelial cells,mRNA sequencing was performed on the cells before and after induction.Differential expression analysis and combined miRNA-mRNA analysis were performed on mRNA sequencing data,and a regulatory network was constructed based on the results of combined miRNA-mRNA analysis.The results of differential gene expression analysis showed that there were 11491 genes with significantly different expression levels before and after transdifferentiation,of which 6,015 were significantly up-regulated and 5629 were down-regulated after transdifferentiation.The results of miRNA-mRNA combined analysis showed that 27 differential target genes and 16 differential miRNAs may be closely related to the differentiation and lactation of transdifferentiated mammary epithelial cells,among which CPT1 B,ECHS1,ACSL1,ACSL6,ACADSB,ALDH3A2,ACOX1,ELOVL3,ELOVL4,ACSF3,SCP2,SCP2,and BCAT2 may have a certain positive regulatory effect on the synthesis and metabolism of milk fat in milk.miR-433,miR-214-3p,miR-29b-3p may have certain regulatory effects on the development of mammary glands and lactation of transdifferentiated mammary epithelial cells,miR-655,miR-671-5p,miR-432-5p,miR-758,miR-455-3p,miR-455-5p,miR-873-5p,miR-7-5p may have certain regulatory effects on the transdifferentiation of goat fibroblasts.In conclusion,this study demonstrated the identity of transdifferentiated mammary epithelial cells at the miRNA level,and established a miRNA-mRNA regulatory network that regulates lactation in transdifferentiated mammary epithelial cells.It was found that PT1 B,ECHS1,ACSL1,ACSL6,ACADSB,ALDH3A2,ACOX1,ELOVL3,ELOVL4,ACSF3,SCP2,SCP2,BCAT2 and miR-433,miR-214-3p,miR-29b-3p,miR-655,miR-671-5p,miR-432-5p,miR-758,miR-455-3p,miR-455-5p,miR-873-5p,miR-7-5p may play a key regulatory role in the differentiation and lactation of transdifferentiated mammary epithelial cells.