Transcriptional Regulation Mechanism Of Bovine PLIN1 Gene And Its Effect On Proliferation,Differentiation And Lipid Metabolism Of Preadipocytes

Lipid droplet coating protein(perilipin,PLIN1)is a protein encoded by the PLIN1 gene in eukaryotes.The PAT family is a family of proteins related to lipid droplet surface proteins.PLIN1 phosphorylation plays a vital role in fat metabolism in adipose tissue and plays an important role in regulating fat breakdown and fat storage in adipocytes.PLIN1 is coated on the outer layer of lipid droplets and prevents lipases from entering the body,such as hormone-sensitive lipase(HSL)and fatty triglyceride lipase(ATGL).The PLIN1 gene has two-way regulation of lipolysis: In the basic state,PLIN1 is coated on the surface of lipid droplets,preventing lipase from contacting triglycerides in the lipid droplets,thereby inhibiting lipolysis.When the energy demand increases,the c AMP-PKA signaling pathway When activated,PLIN1 phosphorylation promotes lipolysis.In order to investigate the regulation of bovine PLIN1 gene on the proliferation and differentiation of bovine adipocytes and lipid metabolism,this study used Qinchuan beef cattle calf precursor adipocytes as experimental materials,through adenovirus-mediated overexpression and interference with PLIN1 gene method,To explore the role of PLIN1 gene in regulating the proliferation and differentiation of bovine precursor adipocytes and lipid metabolism.The main research contents are as follows:1.Correlation analysis of PLIN1 gene polymorphism and growth traits and meat quality traits of Qinchuan beef cattle.The expression of the PLIN1 gene in 12 tissue sites of Qinchuan cattle was detected.The expression of PLIN1 gene in subcutaneous adipose tissue was significantly higher than that of other tissues.In 510 Qinchuan cattle samples,5 SNP loci were detected on the PLIN1 gene by direct sequencing,with distributions g.3579T> C,g.3897G> A,g.8332G> A,g.10516T> C and g.10537G> T.Correlation analysis showed that these five SNP loci were firmly related to some growth and development traits and meat quality traits of Qinchuan cattle.Combination haplotype H2H4 Qinchuan beef quality traits are more likely to provide economic advantages for breeding.2.Transcription factors E2F1,PLAG1,C / EBP? and SMAD3 regulate the transcription of PLIN1 gene.The 5 'end upstream sequence of PLIN1 gene was cloned in 1978 bp,and the core promoter region of the PLIN1 gene was located between-209 and-17 bp by segment deletion and double luciferase activity detection.C / EBP?,E2F1,PLAG1,SMAD3 and other important transcription factors were predicted and screened in the core promoter region.Through site-directed mutagenesis and interference experiments,transcription factors such as C / EBP?,E2F1,PLAG1,and SMAD3 play an important role in maintaining the activity of the PLIN1 gene promoter.Further through EMSA experiments,it was verified in vitro that transcription factors such as C / EBP?,E2F1,PLAG1,and SMAD3 are the key transcription factors of the PLIN1 gene,and it has been verified that the PLIN1 gene has an important regulatory role in bovine adipocytes.3.PLIN1 gene has effects on the proliferation,differentiation and lipid metabolism of bovine precursor adipocytes.The bovine PLIN1 gene adenovirus overexpression and interference vector was constructed and packaged to obtain the over-expressed recombinant adenovirus Ad-PLIN1 and the interfering recombinant adenovirus sh-PLIN1.In bovine precursor adipocytes,overexpression of PLIN1 gene promotes the expression of PLIN1,FASN,PPAR?,ACC,LPL,FABP4,DGAT2,and C / EBP? at the m RNA level,and inhibits the expression of fat metabolism genes such as PLIN2 and ATGL Promoting the expression of genes such as PLIN1,FASN,PPAR?,ACC,LPL,FABP4,and DGAT2 at the protein level inhibits the expression of ATGL gene;similarly,interference with PLIN1 gene in bovine precursor adipocytes will have the opposite result.After overexpression of the PLIN1 gene,oil red O staining results showed that the number of lipid droplets in the fat cells increased and the volume increased,and the triglyceride detection content also increased;after the interference of the PLIN1 gene,the number of lipid droplets in the fat cells decreased,and the volume became smaller The decrease in triglyceride content indicates that the PLIN1 gene can promote the accumulation of triglycerides in bovine precursor adipocytes and has an important regulatory role in fat metabolism.4.Based on transcriptome sequencing analysis of PLIN1 gene on lipid metabolism and key gene mining.Transcriptome sequencing(RNA-seq)was used to analyze the candidate genes of bovine precursor adipocytes treated with Ad-PLIN1 and Ad-NC(no-load virus).Through transcriptome sequencing analysis,a total of 8026 differentially expressed genes were detected.The GO analysis of the differential genes and the analysis of the KEGG signaling pathway were performed.Some of the differential genes were enriched in the AMPK signaling pathway,wnt signaling pathway,and PPAR signaling pathway related to fat proliferation and differentiation.At the transcription level,it shows that the PLIN1 gene has an important regulatory effect on fat proliferation and metabolism.The sequencing results also enriched new differential genes related to fat metabolism pathways,providing theoretical support for molecular breeding of Qinchuan cattle.5.Based on small RNA sequencing analysis of PLIN1 gene on lipid metabolism and key gene mining.To further study the effect of PLIN1 gene on lipid metabolism,small RNA sequencing was performed on transcriptome-sequenced individuals.Analysis of the sequencing results revealed a total of 719 known mi RNAs and 112 new mi RNAs.Thirty-four differentially expressed mi RNAs were found,of which 18 mi RNAs were up-regulated,and 16 mi RNAs were down-regulated.A total of 6000 differentially expressed mi RNA target genes were predicted target genes.GO enrichment analysis,and KEGG pathway analysis was performed on differentially expressed mi RNA target genes,some of which were enriched in fat metabolism,protein metabolism,and cellular immune signaling pathways such as the MAPK signaling pathway,c GMP-PKG signaling pathway,and m TOR signaling pathway.Through the integration analysis of m RNA-seq and small RNA-seq,GO enrichment and KEGG pathway analysis of differentially expressed mi RNA target genes were performed based on the correspondence between mi RNA and its target genes,which were mainly concentrated in the Wnt signal pathway,amino acid biosynthesis,p53 signal pathway,and MAPK signal pathway.In summary,this study conducted a preliminary study on the transcriptional regulation mechanism of bovine PLIN1 gene by constructing a piecewise deletion vector and EMSA experiment,using high-throughput sequencing RNA-seq and small RNA-seq and using multi-omics analysis from m RNA-mi RNA level To study the role of PLIN1 gene in lipid metabolism.