Screening Of Key Genes And LncRNAs In Milk Fat Metabolism And Functional Verification Of PI4K2A In Dairy Cows

Holstein cows with extreme differences in milk lipid rates were screened,and primary dairy mammary epithelial cells（BMECs）were isolated and cultured from fresh milk collected aseptically.Transcriptome combined with weighted gene co-expression network analysis（WGCNA）was used to deeply explore the key candidate genes and lncRNAs that regulate milk fat metabolism in dairy cows and construct a regulatory network of endogenous competitive RNAs（ceRNAs）.Afterwards,the key candidate genes that may regulate milk fat synthesis in dairy cows were validated at the cellular level.The inhibitory effect of trans 10 cis 12 conjugated linoleic acid（t10,c12-CLA）on milk fat synthesis in ruminant livestock is supported by extensive experimental data at both the cellular and nutritional levels,but different scholars have found that it regulates fatty acid translocase（CD36）and long-chain fatty acid coenzyme A ligase 1（ACSL1）in different ways.In this study,we will analyze the regulatory role of t10,c12-CLA on milk fat metabolism at the cellular level and explore whether key candidate genes have an effect on the milk fat inhibition of t10,c12-CLA,which has important theoretical significance and practical value for regulating fatty acid composition and content in milk and improving milk quality,and provides a theoretical basis for understanding the complex biology of milk fat synthesis in dairy cows.The main results of this study are as follows:1.Identification of key lncRNAs and mRNAs for milk fat metabolism in dairy cows by transcriptome and WGCNA technologiesEight Holstein cows with somatic cell counts of 100,000/mL or less and extreme differences in milk fat percentage were selected to successfully isolate and identify BMECs from fresh milk collected aseptically.The results of reverse transcription polymerase chain reaction（RT-PCR）and triglyceride（TAG）kits showed that the expression of milk fat metabolism-related marker genes and TAG content in BMECs of the high milk fat group were significantly higher than those of the low milk fat group,which were fully consistent with the differences in milk fat rate phenotype and milk fat layer.A total of 38 differentially expressed lncRNAs（DELs,padj<0.05）were identified by transcriptome sequencing of high and low milk fat rate BMECs,containing 18 candidate DELs for milk fat metabolism.TCONS₀0082721 is a possible DELs predicted to regulate milk lipid metabolism by co-localization and co-expression together,and its targeting relationship with FABP4 gene needs to be focused on.In addition,we constructed 156 reciprocal regulatory networks of ceRNAs related to milk fat metabolism,and the miR-145-centered regulatory network will be the focus of subsequent studies.Comprehensive analysis of lncRNAs expression profile data using WGCNA revealed that 24 core_lncRNAs were closely associated with lipid metabolism.Based on the endogenous competition mechanism we constructed 22 ceRNAs most likely to be involved in milk lipid metabolism,and TCONS₀0133813 and bta-miR-2454-5p were located at the core of this network,respectively.In addition,transcriptome sequencing identified a total of 47 differential genes（DEGs,padj<0.05）.Eleven candidate DEGs were screened by functional enrichment analysis for possible regulation of milk fat metabolism,namely PDGFD,BCAT1,APOL3,ATP8A2,PTPRR,KCNMA1,ZFYVE28,ENPP2,DKK1,CES4A and CTSH.A comprehensive analysis of mRNA expression profile data using WGCNA screened a total of five candidate DEGs related to milk fat metabolism,namely PI4K2A,SLC16A1,ATP8A2,VEGFD and ID1,of which ATP8A2 was the candidate DEG obtained by transcriptome and WGCNA together.2.Identification and functional analysis of key candidate genes for milk fat metabolismTissue expression profiling of 15 candidate DEGs for milk lipid metabolism obtained by transcriptome and WGCNA analysis revealed that the expression levels of ENPP2,PI4K2A,CTSH and PTPRR genes were higher in breast tissues than in other tissues.Relative to other genes,PI4K2A and CTSH were expressed at higher levels in both breast tissues and BMECs.PI4K2A was identified as a key candidate gene for regulating milk fat synthesis in dairy cows,and the results of nucleoplasmic isolation experiments showed that it was mainly expressed in the cytoplasm The CDS region of PI4K2A gene is 1440 bp long and encodes 479 amino acids.The functional prediction revealed that PI4K2A is a non-secretory and unstable hydrophilic protein with many phosphorylation sites and abundant post-translational modifications.the secondary and tertiary structures of PI4K2A protein are mainly irregularly coiled,followed by α-helix.3.Effect of PI4K2A gene on milk fat synthesis and proliferation and apoptosis of BMECs in dairy cowsThe results of overexpression and interference experiments showed that the PI4K2A gene positively regulated fatty acid transport processes in dairy cows’ milk lipid synthesis and indirectly regulated fatty acid de novo synthesis,long-chain fatty acid（LCFA）uptake and fatty acid（FA）desaturation.The PI4K2A gene was also found to have a positive regulatory effect on milk fat synthesis in dairy cows by examining TAG,total cholesterol content and lipid droplet secretion in BMECs.In addition,the inhibitory effect of interfering with PI4K2A on milk fat may be stronger than the promoting effect of overexpressing PI4K2A on milk fat synthesis,which laterally reflects that PI4K2A gene may be an important regulator dependent on milk fat synthesis process in cows.The results of EdU and apoptosis kit assays showed that overexpression of PI4K2A gene promoted the proliferation and inhibited the apoptosis of BMECs;on the contrary,interference with P14K2A gene inhibited the proliferation and promoted the apoptosis of BMECs.4.PI4K2A mediates the role of t10,c12-CLA in regulating milk fat inhibition in dairy cowsExogenous addition of t10,c12-CLA at 170 μM to BMECs cultured in vitro significantly inhibited FA de novo synthesis,LCFA uptake,transport and desaturation,and TAG synthesis and lipid droplet secretion in dairy cows,and this inhibition may be potentially and importantly linked to the downregulation of PPARs,ELOVL6 and EGFR genes.Overexpression of P14K2A attenuated the inhibitory effect of t10,c12-CLA on milk fat,and knockdown of PI4K2A had no significant effect on the milk fat inhibitory effect of t10,c12-CLA,suggesting that the inhibition of milk fat synthesis in cows by t10,c12-CLA may not depend on PI4K2A.In summary,this study identified 15 candidate differential genes and 18 DELs related to milk fat metabolism by transcriptomics and WGCNA analysis of BMECs,and finally identified PI4K2A as a key candidate gene regulating milk fat metabolism by combining qRT-PCR techniques.The PI4K2A gene positively regulated milk fat synthesis and proliferation of BMECs,and negatively regulated apoptosis of BMECs in dairy cows.PI4K2A gene overexpression attenuated the inhibitory effect of t10,c12-CLA on milk fat.The results of this study provide a basis for the analysis of the regulatory mechanism of milk fat traits and provide genetic marker information for precise molecular breeding of milk composition traits in dairy cows.