| The mammary gland is a unique exocrine gland.It is one of the few organs in mammals that can repeatedly undergo the process of growth,functional differentiation and degradation.Mammary gland as a production organ of mammals can convert nutrient elements into milk components and then form milk.Milk is a metabolite of the physiological activities of lactation.It contains nutrients necessary for the growth and development of the newborn pups.It is an ideal food for feeding newborn offspring.Bovine milk is secreted by bovine mammary epithelial cells(BMECs),and it contain protein,carbohydrates,lipids,water and ions.For many years,how the various components of milk coordinate and secrete in the cell has been plagued by cell biologists and lactation biologists.In recent years,with the changes in human nutrition concepts,low-fat,high-protein milk has become the main market demand.Therefore,producing milk that meets market demand can not only improve the economic benefits of milk manufacturers,but also benefit human health.Only a thorough understanding of the gene metabolism mode and regulation mechanism in mammary glands can reasonably regulate the synthesis and secretion of milk components such as milk fat and protein.With the rapid development of sequencing technology and bioinformatics,the advent and application of high-throughput sequencing and various omics technologies have promoted unprecedented progress in the field of mammary cell function.This subject is based on Chinese Holstein dairy cows.First,the changes in the main milk components are measured and analyzed during a lactation cycle(7 days before calving,-7 d;30 days after calving,30 d;90 days after calving,90 d;180 days after calving,180 d;270 days after calving,270 d;305 days after calving,305 d);Then,differentially expressed genes(DEGs),differentially expressed microRNAs(DE miRNAs)and differential signaling pathways were identified at different stages of breast development using RNA-seq,miRNA-seq technology and bioinformatics analysis methods;In addition,DE miRNAs and DEGs pairs with the potential targeting relationships were predicted,in which pairs potentially related to fatty acid metabolism were screened according to the change in the main milk components.Finally,the molecular mechanism of DE miRNA and DEGs pairs(miR-193a-5p and FADS1,miR-378d and FADS2,miR-128 and ELOVL6)was explored in milk fatty acid metabolism of BMECs.The results of the study are as follows:(1)The RNA-seq data from adjacent time nodes showed that there were 2421 DEGs in the-7 d vs.30 d group(P?0.05),of which 1073 DEGs were up-regulated and 1348 DEGs were down-regulated.There are 33 DEGs in the 30 d vs.90 d group(P?0.05),of which 15 DEGs are up-regulated and 18 DEGs are down-regulated;there are 313 DEGs in the 90 d vs.180 d(P?0.05),of which 206 DEGs are up-regulated and 107 DEGs are down-regulated;the 180 d vs.270 d group is present 718 DEGs(P?0.05),of which 293 DEGs were up-regulated and 425 DEGs were down-regulated;there were 1207 DEGs(P? 0.05)in the 270 d vs.305 d group,of which 828 DEGs were up-regulated and 379 DEGs were down-regulated.Studies have found that drastic and complex regulation of gene expression is carried out in mammary gland cells with the advancement of lactation.(2)Based on the RNA-seq data of mammary gland samples from all periods,this experiment for the first time identified 10 significant modules related to lactation period,milk yield and main milk component using weighted gene co-expression network(WGCNA)analysis.DEGs in the 10 specific modules were performed to Genetic Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The results showed that the galactose metabolic pathway is a potential candidate pathway for milk yield and lactose synthesis.Intracellular ion transport is more frequent and cell proliferation becomes active at the beginning of lactation.In late lactation,suppressor of cytokine signaling 3(SOCS3)may be involved in cell apoptosis.Sphingolipid signaling pathway is a potential candidate pathway for milk fat synthesis.(3)The miRNA-seq data at adjacent time nodes showed that there were 45 DE miRNAs in the-7 d vs.30 d group(P?0.05),of which 31 miRNAs were up-regulated and 14 miRNAs were down-regulated;there were 13 DE miRNAs in the 30 d vs,180 d group(P?0.05),of which 6 miRNAs were up-regulated and 7 miRNAs were down-regulated;there were 19 DE miRNAs in the 180 d vs.305 d group(P?0.05),of which 7 miRNAs were up-regulated and 12 miRNAs were down-regulated.(4)The interaction network of DE miRNA-DEGs expression in mammary gland was constructed according to the data of DEGs and DE miRNAs pairs in different periods.Potential pairs related to fatty acid metabolism were identified.Three pairs,miR-193a-5p-FADS1,miR-378d-FADS2 and miR-128-ELOVL6,were applied for subsequent research.(5)The dual luciferase reporter assay,qRT-RCR,Western blotting,triglyceride(TAG)assay and fatty acid analysis were utilized for exploration the function of miR-193a-5p and its potential target gene,FADS1.The dual luciferase reporter indicated that miR-193a-5p in BMECs targeted to the 3'UTR region of FADS1,thereby regulating its expression level.The results of qRT-PCR and Western blotting displayed that FADS1 expression in BMECs was negatively correlated with the miR-193a-5p expression,regardless based on mRNA levels or protein levels.The content of TAGs,eicosapentaenoic acid(EPA),docosapentaenoic acid(DPA)and docosahexaenoic acid(DHA)were positively correlated with FADS1 expression in BMECs(P?0.05),and negatively correlated with the expression of miR-193a-5p(P ?0.05).This study demonstrated for the first time that miR-193A-5p regulates the metabolism of polyunsaturated fatty acids(PUFAs)in BMECs by targeting FADS1,suggesting that miR-193A-5p and FADS1 are potential factors for increasing PUFAs content in bovine milk.(6)The dual luciferase reporter assay,qRT-RCR,Western blotting,TAG content assay and fatty acid analysis were used to explore the function of miR-378d and its potential target gene,FADS2.The dual luciferase reporter revealed that miR-378d in BMECs targeted to the 3'UTR region of FADS2,thereby regulating its expression level.The data of qRT-PCR and Western blotting showed that FADS2 expression in BMECs was negatively correlated with the miR-378 expression levels on both mRNA and protein levels.The contents of TAG,arachidonic acid(AA)and DHA were significantly positively correlated with the expression of FADS2(P?0.05),and significantly negatively correlated with the expression of miR-378d(P?0.05).This study demonstrated for the first time that miRNA-378d regulates the metabolism of PUFAs in BMECs by targeting FADS2,suggesting that miR-378d and FADS2 are potential factors for increasing PUFAs content in bovine milk.(7)The dual luciferase reporter assay,qRT-RCR,Western blotting,TAG content assay and fatty acid analysis were used to explore the function of miR-128 and its potential target gene,ELOVL6.The dual luciferase reporter proved that ELOVL6 is the target gene of miR-128 in BMECs.qRT-PCR and Western blotting manifested that ELOVL6 expression in BMECs was negatively correlated with the miR-128 expression levels on both mRNA and protein levels.The contents of TAGs,stearic acid(SA)and oleic acid(OA)were significantly positively correlated with the expression of ELOVL6(P?0.05),and significantly negatively correlated with the expression of miR-128(P?0.05).This study was the first to confirm that miR-128 regulates the fatty acid metabolism in BMECs by targeting ELOVL6,indicating that miR-128 and ELOVL6 are potential factors to increase the content of long chain fatty acids(LCFAs)in milk.In summary,this study revealed the differential expression of genes and miRNAs in the bovine mammary glands at different stages of lactation.This work verified the target relationship between the identified DE miRNAs and DEGs,and clarified the functions of three DE miRNAs-DEGs pairs in BMECs.The current study made contributions to intimate the mechanism of lipid synthesis and metabolism,realize the directional genetic regulation of milk components,and lay the theoretical foundation to produce high-quality raw milk. |
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