Regulatory Mechanisms And Function Study On Lipid Metabolism Of VNN1 Gene In Chicken

Vanin-1(VNN1)is a pantetheinylamine that catalyzes the hydrolysis of panthenylethylamine to produce pantothenic acid(vitamin B5)and mercaptoethylamine(highly effective antioxidant).Studies in mammals have shown that the VNN1 gene plays a very important role in life processes such as glycolipid metabolism,immunity,and tumor formation.Our previous study found that this gene is specifically expressed in chicken liver and is negatively regulated by microRNA-122,a key regulator of liver lipid metabolism.However,to date,studies on the chicken VNN1 gene have been reported less.So it is necessary to conduct an in-depth study on the regulation of chicken VNN1 gene expression and its function in liver lipid metabolism.In this study,the chicken VNN1 gene was used as a research object to clarify the specific mechanism of chicken VNN1 expression regulation and its role in liver lipid metabolism.First,this study used CRISPR/Cas9 knockout technology to knock out the VNN1 gene in chicken liver cell LMH,and analyzed the differential expression of lipid metabolism-related genes caused by the deletion of VNN1 gene by transcriptome sequencing,thereby analyzing VNN1.The function of genes in lipid metabolism in chicken liver.Secondly,the transcriptional regulation mechanism of VNN1 gene was demonstrated by bioinformatics analysis,luciferase reporter gene assay,site-directed mutagenesis and transcription factor PPARa and HNF4α function acquisition and deletion experiments.Finally,by studying the pairing of the 3’-UTR region of the VNN1 gene with the microRNA,a microRNA capable of pairing with the VNN1 gene was screened and verified by the dual luciferase reporter gene to research the regulation mechanism of VNN1.This article will be explained in the following five sections:(1)In order to explore the application of CRISPR/Cas9 system in chicken-derived hepatocytes,this study used LMH as a targeting cell and transfected the constructed pX330-VNN1-sgRNA1/2/3 knockout plasmid into LMH cells.The genomic DNA of the cells was extracted after preliminary screening of puromycin,and the knockout efficiency was detected by T7E1 enzyme digestion and TA cloning.The results showed that the CRISPR/sgRNA3 system can effectively achieve the targeted mutation of VNN1 gene in chicken LMH cells,and the knockout efficiency of TA clone was up to 38.7%.Next,in this study,pX330-VNN1-sgRNA3 transfected LMH cells were enriched by multi-round puromycin,and the enriched cellular RNA was extracted for real-time PCR analysis.The results showed that the expression level of VNN1 mRNA in LMH-VNN1-/-cells was decreased by about 80%compared with wild-type LMH cells(P<0.0001).The above results indicate that this experiment has successfully established a CRISPR/Cas9 system that can effectively achieve gene-targeted modification in chicken LMH cells.(2)In order to study the specific function of VNN1 in chicken fat metabolism,this study used the Illumina HiSeq sequencing platform for paired-end(PE)sequencing of the VNN1 gene in LMH cells.Sequencing results showed that 1335 genes were differentially expressed in LMH cells due to defects in VNN1 gene,of which 431 genes rose and 904 genes decreased.The enrichment analysis of differential gene GO showed that the related lipid metabolism pathways,such as regulation of fatty acid biosynthesis and fatty acid metabolism,long-chain fatty acid biosynthesis processes were enriched;KEGG metabolic pathway enrichment analysis also showed pantothenic acid and CoA biosynthesis;Oil metabolism;biosynthesis of unsaturated fatty acids;fatty acid biosynthesis and other pathways are enriched.Screening of the above-mentioned enrichment pathway DEGs revealed a total of 76 genes with significant differences,of which 29 genes rose and 47 genes decreased,indicating that VNN1 plays an important role in coordinating chicken liver lipid metabolism.(3)In this study,we first used the chicken starvation-supplementation model to explore the expression of VNN1 and related glycolipid metabolism genes under the condition of nutritional status changes.The results showed that VNN1 gene was similar to FASN/ATGL/ACOX1 and other genes.Regulation,and the expression of the transcription factors PPARa and VNN1 genes are strongly consistent;the use of PPARa agonist GW7647 and PPARa to interfere with siRNA treatment of hepatocytes,showing that PPARa has a positive regulation of VNN1 gene expression;subsequent bioinformatics analysis,Luciferase reporter assay and site mutation analysis revealed that PPARa upregulates the transcriptional activity of VNN1 by binding to the PPARa binding site of the VNN1 gene promoter-49/-31 region.This part preliminarily showed that the chicken VNN1 gene is positively regulated by the transcription factor PPARa,which provides a theoretical basis for further study of the specific mechanism of PPARα-VNN1 axis on chicken liver lipid metabolism.(4)Based on the codon preference of E.coli,this study optimized the HNF4a gene codon and successfully constructed the prokaryotic expression vector pET-3 0a(+)-HNF4α of HNF4a.The protein was induced by IPTG and purified by Ni2+-NTA agarose affinity chromatography to obtain a higher purity HNF4α protein.The rabbit anti-chicken HNF4αpolyclonal antibody with high titer and specificity was successfully prepared by using purified HNF4α protein as immunogen,and the antibody could better characterize the protein level of HNF4α overexpression and interference.The acquisition and deletion of HNF4α significantly affected the expression of VNN1 gene.This result indicates that HNF4α plays an important role in the expression of VNN1 gene,which provides basic information for further understanding of the function of chicken HNF4α-VNN1 axis.(5)Using the miRanda(a miRNA prediction software)to predict the microRNAs targeting VNN1 gene,in the candidate microRNAs,four microRNAs with score>160 and free energy<-15 kCal/Mol were selected for in vitro cell validation.Through the above bioinformatics and dual luciferase assays,gga-miR-181a-5p can target the regulation of VNN1 gene.This also indicates to a certain extent that the VNN1 gene is regulated by upstream gga-miR-181a-5p in regulating chicken lipid metabolism,enriching the specific mechanism of chicken VNN1 gene regulation at post-transcriptional level.