Molecular Mechanisms Of Long Noncoding RNA-lncHLEF Regulating Hepatic Lipid Metabolism And Intramuscular Fat Deposition In Chicken

In chicken,the main metabolic organ for lipids is the liver,where more than 90% of the de novo fatty acids are synthesized.Although the synthesis and transport of chicken liver lipids has always been the focus of research in poultry field,the molecular mechanism of its regulation is still unclear.Long noncoding RNAs(lncRNAs)are a class of non-coding RNA molecules with a length greater than 200nucleotides(nt)that play a role in the regulation of various biological processes.However,the study of their roles in chicken liver lipid metabolism and their molecular mechanisms is still in its infancy.Due to the physiological characteristics of egg-laying in birds,oocytes must store all the necessary nutrients for embryonic development,and almost all lipid substances are synthesized in the liver and transported to developing oocytes in the form of lipoproteins,while laying hen’s ovaries do not synthesize lipids.Therefore,the period of egg production is a particularly active period for liver lipid metabolism.It is precisely because of this characteristic that laying hens during the pre-laying and peak-laying period become an ideal model for studying liver lipid metabolism in chickens.In this study,the pre-(20-week)and peak-laying(30-week)Lushi blue-shelled-egg chickens were used as the experimental subjects,and Oxford Nanopore Technologies(ONT)third-generation sequencing technology was used to screen differentially expressed lncRNAs(DE-lncRNAs)and genes(DEGs)during two periods.Predict the cis-and trans-regulatory target genes of newly identified differentially expressed lncRNAs,and construct a(DE-lncRNA-DEGs)interaction network to identify the key lncRNA lncHLEF for regulating liver lipid metabolism.In vivo experiments,combined with in vitro gain-and loss-of-function experiments,were conducted to determine the biological function of lncHLEF in chicken liver lipid metabolism.Subcellular localization of lncHLEF,along with RNA pulldown technology and bioinformatics analysis,was employed to screen potential interacting micro RNAs(miRNAs)with lncHLEF.A core interaction network was constructed,considering lncHLEF as a competing endogenous RNA(ceRNA),to explore the ceRNA molecular mechanism of lncHLEF in targeted regulation of chicken liver lipid metabolism.Potential coding peptide ability analysis of lncHLEF was performed to elucidate the function and molecular mechanism of the encoded peptide in liver lipid metabolism.Furthermore,co-culture experiments of exosomes and primary preadipocytes were conducted to investigate the impact of liver-derived exosomes-mediated lncHLEF on lipid deposition in intramuscular and abdominal preadipocytes.The main results obtained are as follows:Part Ⅰ Identification of key full-length lncRNAs of liver lipid metabolism through thirdgeneration transcriptome sequencingThrough ONT sequencing of the liver samples from 20-and 30-week-old LS hens,a total of 1,106 differentially expressed transcripts were identified,including 391 DE-lncRNAs and 537 DEGs,among which 68 DE-lncRNAs and 99 DEGs were newly identified.The cis-and trans-acting target genes of these new DE-lncRNAs were predicted by genome location relationship and LncTar software,and the target genes enriched in lipid metabolism-related pathways and differentially expressed were screened.After sorting the top ten(Top 10)DE-lncRNAs with the most significant differential expression during the pre-laying and peak laying periods according to the False Discovery Rate(FDR),it was found that the target gene of the top 2 lncRNA PB1646.2 was the transcription factor GATA binding protein 6(GATA6),which has multiple biological functions.Therefore,it was hypothesized that lncRNA PB1646.2 might play a key role in the regulatory network of lipid metabolism,and it was named lncHLEF(lncRNA hepatic lipogenesis enhancement factor)for subsequent research.Part Ⅱ Study on biological function of lncHLEF in liver lipid metabolismReal-time fluorescence quantitative PCR(qPCR)analysis showed that lncHLEF was differentially highly expressed during the peak laying period.The full-length sequence of 1,350 nt lncHLEF was successfully amplified by PCR,and the result was consistent with the ONT sequencing data.RNA FISH and nuclear-cytoplasmic fractionation experiments indicated that lncHLEF was predominantly expressed in the cytoplasm.In vitro and in vivo functional tests showed that lncHLEF could significantly promote the expression of lipid synthesis and transport genes in hepatocytes,activate the expression of triglyceride(TG)and total cholesterol(T-CHO)synthesis pathway genes,and promote lipid synthesis in hepatocytes.Part Ⅲ Molecular mechanism of lncHLEF regulating liver lipid metabolism through ceRNA networkRNA pull-down-seq and bioinformatics analysis identified miRNA-2188-3p as a shared target miRNA for lncHLEF and GATA6,and dual-luciferase reporter assays and rescue experiments demonstrated that miRNA-2188-3p could directly interact with lncHLEF and GATA6.In vivo and in vitro functional experiments revealed that GATA6 promoted the expression of lipid synthesis and transport genes in hepatocytes,as well as intracellular lipid synthesis,while miRNA-2188-3p inhibited the expression of these genes and intracellular lipid synthesis in hepatic cells.LncHLEF acted as a competitive endogenous RNA to sequester miRNA-2188-3p,thereby increasing the expression of the miRNA-2188-3p target gene GATA6 and promoting hepatic lipid metabolism in chicken.Part Ⅳ Molecular mechanism of lncHLEF regulating liver lipid metabolism by encoding peptidesBioinformatics analysis showed that the lncHLEF sequence contains three potential open reading frames(ORFs),ORF2,ORF3 and ORF4.In vitro functional assays demonstrated that ORF2/3/4 could promote lipid synthesis,expression of transport genes and intracellular lipid synthesis in hepatocytes.Using Co-IP/LC-MS/MS,1,251,878 and 1,297 proteins were screened for peptide binding to ORF2,ORF3 and ORF4,respectively.Functional enrichment analysis revealed that the peptide-interacting proteins were significantly enriched in pathways related to lipid intake,transport,synthesis,and degradation.Cell immunofluorescence and IP experiments demonstrated the cytoplasmic co-localization of the ATP citrate lyase(ACLY)protein with peptides ORF2,ORF3,and ORF4,and ACLY protein may target and bind to these three peptides.Further analysis using Alpha Fold and ZDOCK software showed that ACLY protein and peptides ORF2,ORF3 and ORF4 had 7,2 and 6 amino acid binding sites,respectively.Mutation experiment of the binding sites,CHX chase,and Western blot experiments revealed that the three encoded peptides maintained the stability of the ACLY protein,attenuated its degradation,and promoted the expression of genes involved in TG and cholesterol(CHO)pathways,thereby facilitating hepatic lipid metabolism in chicken.Finally,the loss of lncHLEF encoding functionality and rescue experiments demonstrated that lncHLEF can promote hepatic lipid synthesis through two mutually independent mechanisms of action,mediating the miR-2188-3p/GATA6 axis and encoding endogenous peptides,respectively.Part Ⅴ Molecular mechanism of lncHLEF regulating fat differential deposition through exosomesAnalysis of Oil Red O and H&E staining results of muscle tissue sections from individuals tested in vivo for liver-specific expression of lncHLEF adeno-associated viral vector(AAV-lncHLEF)showed that lncHLEF promotes intramuscular fat deposition without affecting abdominal fat deposition.In vitro functional experiments demonstrated that lncHLEF enhanced the expression of genes associated with intramuscular fat synthesis in intramuscular preadipocytes(IMF-pre)and intracellular TG synthesis but had no effect on lipid deposition in abdominal fat preadipocytes(Ab F-pre).qPCR and PCR analysis of individual plasma and in vitro cultured hepatocyte exosomes revealed the presence of lncHLEF in liverderived exosomes.Co-culture experiments of liver-derived exosomes with primary adipocytes demonstrated the transfer of lncHLEF from liver-derived exosomes to target adipocytes,promoting the expression of genes related to intramuscular fat synthesis in IMF-pre cells and intracellular TG synthesis,while having no effect on lipid deposition in Ab F-pre cells.The experiments conducted above demonstrate that lncHLEF not only significantly enhances hepatic lipid synthesis but also markedly increases intramuscular fat deposition.In summary,in this study,391 DE-lncRNAs and 537 DEGs were identified in the livers of Lushi blue-shelled-egg chicken during the pre-and peak-laying periods by using ONT sequencing technology,and the regulatory network of hepatic lipid metabolism in chickens was constructed,and the in vivo and in vitro functional assays were performed to further validate the regulatory function of lncHLEF,a key regulatory lncRNA in the network,in hepatic lipid metabolism.Using the in vitro gain-and loss-offunction strategy,combined with bioinformatics analysis and molecular biological techniques,we elucidated that lncHLEF can regulate hepatic lipid metabolism through both the ceRNA network and the encoded endogenous peptide,as well as the molecular mechanism by which hepatic-derived exosomes mediate the regulation of differential lipid deposition by lncHLEF.This research will further enrich the theory of hepatic lipid metabolism regulation in chickens(poultry),achieve targeted regulation of hepatic lipid metabolism,and provide theoretical support for the selection and breeding programs aimed at improving the productive performance and the quality of meat and eggs in chickens(poultry).