| More and more evidences proved that,except the traditional factors influence the lipid metabolism,some new discovered regulatory factors,such as noncoding RNA（ncRNA）including miRNA and lncRNA also play an important role in regulating lipid metabolism.Some related enzymes in mammals playing important roles in fat synthesis and lipoprotein assembly do not have the same function in chicken and other birds.Therefore,the molecular regulatory mechanisms of lipid metabolism remain unclear in in egg-laying chicken.In this study,the pre-（20-week）and peak-laying（30-week）Lushi green-shelled hens were used as the experimental model,and RNA-Seq technology was used to investigate the mRNA,miRNA and lncRNA expression profiles of liver tissue,and identify the differentially expressed（DE）mRNAs,miRNAs and IncRNAs.Bioinformatics methods were used to predict the target genes of DE miRNAs and IncRNAs.Gene ontology（GO）annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis were conducted to the DE genes and DE target genes of miRNAs and lncRNAs,respectively.Through integrated analysis of multiple omics,the key genes may directly involve in regulating the lipid metabolism were screened through establishing the regulating network of hepatic lipid metabolism.At the same time,the expression regulation characteristics of the selected genes were validated in vitro and in vivo through animal and cell models.The main results are as follows:Part Ⅰ.Hepatic lipid metabolism related genes in egg-laying chickenThe data statistical analysis of mRNA expression profile showed that,compared with pre-laying chicken,2567 differentially expressed genes（P<0.05）,and 1767 novel genes were obtained in the liver of peak-laying chicken.Among them,960 known and 161 novel genes were considered as significant differentially expressed（SDE）with a false discovery rate（FDR）of ≤0.05 and fold-change ≥ 2 or ≤ 0.5.KEGG pathway analysis showed that the SDE genes were significantly enrichment in steroid biosynthesis,PPAR signaling pathway,biosynthesis of unsaturated fatty acids,glycerophospholipid metabolism,pyruvate metabolism and so on（P≤0.05）.GO annotation analysis showed that the SDE genes were significantly enrichment in lipid biosynthesis,transport and location;cholesterol and sterol metabolic,immune response,and some other terms（P≤0.05）.It is worth noting that the expression of some genes which play crucial roles in mammal lipid metabolism were not altered as expected in mammal,such as MTTP and DGAT2 were not significantly changed between the two periods,and LPGAT1 was significantly down-regulated.in peak-laying stage.The results suggested that there are obvious difference among the genes involved in hepatic lipid metabolism between mammal and chicken.Part II The expression regulation of PNPLA3 gene in chicken liverPNPLA3 protein including a patatin domain has the activity of hydrolysis phospholipids and neutral lipid,and ester acyltransferase activity.PNPLA3 was significantly up-regulated in peak-laying chicken liver,but the expression regulation was unclear.The spatio-temporal expression analysis of PNPLA3 in Lushi green-shelled chicken was conducted by using qRT-PCR technology,and showed that PNPLA3 was highly expressed in breast muscle tissue at 10-week,and in liver at 30-week;PNPLA3 was significant highly expressed in liver at 30-and 35-week than 20-week and before（P≤0.05）.The expression of PNPLA3 presented dose dependent increase when 10-week-old chickens treated with different concentrations of estradiol for 24h,and with the highest expression when treated with the concentration of 2.0 mg-/kg.The expression of PNPLA3 showed dose dependent when CEH stimulated with different concentrations of estradiol for 24h,and significantly higher in 50nM and 100nM groups than the others（P ≤ 0.05）.Moreover,antagonist of estrogen receptors treated hepatocytes showed that the regulation of estrogen on the expression of PNPLA3 may be mediated by ER-a.Part III Hepatic lipid metabolism regulation related miRNAs in egg-laying chickenTotal of 565 known miRNAs were identified in the miRNA expression profile of chicken liver tissue.425 miRNAs were commonly expressed in both stages,46 was only expressed in pre-laying chicken,and 94 only expressed in peak-laying.Compared with pre-laying stage,67 down-and 13 up-regulated miRNAs were verified to be SDE miRNAs（false discovery rate,FDR≤0.05）at the peak-laying stage.MiR-22-3p and miR-146b-5p showed the most abundant expression and the highest fold-change（-8.5）,respectively.Besides,19 DE novel miRNAs（P≤0.05）were identified at peak-laying period.At the same time,the target genes of 80 SDEs were predicted and DE target genes were found among mRNA expression profile.GO enrichment analysis demonstrated that DE target genes were mainly involved in the lipid biosynthetic process,phospholipid biosynthetic and metabolic,fatty acid biosynthetic and metabolic,organic acid biosynthetic,regulation of protein kinase cascade,and phosphate metabolic processes;mitochondrion,endoplasmic reticulum and phosphoinositide 3-kinase complex cell component;and small GTPase regulator activity,protein tyrosine phosphatase and phosphatase activity,phosphotransferase activity and so on（P ≤ 0.05）.KEGG enrichment analysis showed that DE putative target genes were significantly enriched in steroid biosynthesis,glycerophospholipid metabolism,biosynthesis of unsaturated fatty acids,pantothenate and CoA biosynthesis,and PPAR signaling pathway.Moreover,the integrated analysis of miRNA and mRNA indicated that some of the SDE miRNAs might serve as the important regulatory factors in chicken hepatic lipid metabolism process.Part IV ELOVL6 the target gene of miR-22-3pMiR-22-3p was abundant and significantly expressed in chicken liver between pre-and peak-laying groups.Several bioinformatics methods were used to predict the targets of miR-22-3p and a potential target gene of ELVOL6 was selected.The conservation ofmiR-22-3p and the binding sites between miR-22-3p and ELVOL6 3’UTR in variousspecies were analyzed,and showed both of them were highly conserved among species.QRT-PCR analysis to the expression of miR-22-3p and ELVOL6 in different tissues of 10-week-old chickens and in liver at different stages showed miR-22-3p was widely expressed in different tissues,and ELVOL6 was relative highly expressed in liver and kidney;the expression of miR-22-3p in liver showed a rise in early development,but significantly decreased after 15-week（P ≤ 0.05）;ELVOL6 was significantly higher expressed at 30-week than the other periods（P ≤ 0.05）.miR-22-3p mimics and ELVOL6 3’UTR wild-type plasmid or mutant-type plasmid were used to co-transfected DF1 cells,respectively,the luciferase activity was decreased when miR-22-3p mimics and ELVOL6 3’UTR wild type recombinant vector co-transfected the cells;qRT-PCR analysis showed that miR-22-3p mimics led to a significant increase of miR-22-3p and significantly decreased the expression of endogenous ELVOL6 in the CEH（P≤0.05）.Thus it was suggested that miR-22-3p may regulate the fatty acid chain elongation by targeting ELOVL6.PartV Hepatic lipid metabolism regulation related lncRNAs in egg-laying chickenThe analysis of IncRNA expression profile showed a total of 1730 DE lncRNAs wereidentified,including 561 known and 1169 novel IncRNAs.Compared with pre-laying stage,total of 68 lncRNAs were SDE in peak-laying stage,including 62 known and 6 newly discovered.The target gene of 68 SDE lncRNAs was predicted and 315 potential target genes were obtained.Function annotation analysis showed that the target genes mainly enriched in arachidonic acid secretion,regulation of phospholipase C activity,icosanoid secretion and transport,fatty acid derivative transport,monocarboxylic and long-chain fatty acid transport processes（P<0.01）;biosynthesis of unsaturated fatty acid,and a-linolenic acid metabolism pathway（P:≤ 0.05）.It could be inferred that the DE IncRNAs might play important roles in liver metabolism process.The integrated analysis among mRNA-lncRNA-miRNA was conducted,and suggested that there were 13 common potential target genes between the SDE IncRNAs and miRNAs.It was suggested that miRNAs and IncRNAs as the endogenous regulatory factors might be involved in regulating the complex physiological processes of hepatic lipid metabolism in chicken.Part VI Tissue distribution of IncLER and its expression and regulation mechanism in chicken liverRNA-Seq result showed that IncLER（TCONS00037753）was highly expressed in liver of laying chicken,but almost undetectable in pre-laying chicken liver.To better understand the biological function of IncLER in chicken liver,the tissues expression pattern,spatio-temporal expression,and the expression regulation mechanism in estrogen treated individuals and cells models were studied.The results showed that lncLER was extensively expressed in tissues;and its expression in the liver of peak-laying chicken（30w and 35w）is significantly higher than the pre-laying chicken（15w and 20w）（P<0.05）.The expression of IncLER presents an estrogen dose-dependent in chicken liver tissue.The expression level in 2mg/kg and 8mg/kg treated groups was significantly higher than 0.5mg/kg and control groups（P ≤ 0.05）.The expression of IncLER also showed a dose-dependent in CEH treated with different concentrations of estrogen.LncLER was extensively expressed among tissues;its expression was estrogen dose-dependent,and significantly higher in the liver of peak-laying chicken than the pre-laying.LncLER might be involved in the lipid metabolism induced by estrogen.In conclusions,our results suggested that there are obvious difference among the genes involved in hepatic lipid metabolism between mammal and chicken;SDE PNPLA3 could be induced by estrogen and mediated by ER-a;SDE miRNAs might serve as the important regulatory factors in chicken hepatic lipid metabolism process;High abundant SDE miR-22-3p may regulate the fatty acid chain elongation by targeting ELOVL6;some SDE IncRNAs might be involved in regulating the complex physiological processes of hepatic lipid metabolism in chicken.SDE LncLER might be involved in the lipid metabolism induced by estrogen.This study enriched the chicken liver lipid metabolism（poultry）theory,provided a foundation for revealing the molecular mechanism of hepatic lipid synthesis,assembly and transfer,and the application in poultry breeding. |
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