| BackgroundIn recent years,the morbidity and mortality of arteriosclerotic coronary artery disease（CAD）remain high in China,and abnormal blood lipid metabolism is an independent risk factor of atherosclerosis（AS）.Thus,blood lipid control is of great significance for the prevention and treatment of CAD.The etiology of abnormal blood lipid metabolism is complicated and affected by both genetic and environmental factors while genetic factors play an important role in the pathogenesis of lipid metabolism.Among them,single nucleotide polymorphism（SNP）is the most common human heritable variation.In genetic analysis,SNP has been widely used as a kind of genetic marker because’ of its wide-distribution and high genetic stability.By studying the relationship between DNA polymorphic markers which represented by SNP and dyslipidemia and CAD,we can identify the related susceptible genes,and then clarify the relationship between genotype and phenotype,which is helpful to understand the genetic mechanism of disease and provide individual clinical treatment.At present,large-scale genome-wide association studies（GWAS）have reported more than 400 loci affecting blood lipid levels.Previous study has found that there was a significant association between endonuclease exonuclease phosphatase family domain containing-1（EEPD1）gene rs4302748 locus and low-density lipoprotein-cholesterol（LDL-c）level（P=2.10×10-8）in including 500,000 Chinese-European cross-ethnic individuals in exome array study.EEPD1 gene is an endonuclease at the 5 ’end of DNA,which participates in DNA repair and promotes the restart of stress replication forks at DNA double-strand breaks.At the same time,EEPD1 inhibits DNA damage repair through classical non-homologous end junctions and trace homologous-mediated end junctions.Previous study has reported that EEPD1 could promote cholesterol efflux from macrophages by activating adenosine triphosphate binding cassette transporter A1（ABCA1）,suggesting that EEPD1 might play an important role in the regulation of lipid metabolism.ObjectiveBased on the evidence of human genome studies and functional research,this study aimed to clarify the mechanism of EEPD1 gene in regulating blood lipid metabolism in vivo and in vitro,and to provide scientific evidence and potential intervention targets for the prevention and treatment of lipid metabolic disorders and CAD in the future.MethodsThis study was conducted with the combination of population data,cellular research and animal models,mainly using quantitative polymerase chain reaction（qPCR）,Western blotting（WB）and CRISPR-Cas9 technology.1.The effect and molecular mechanism of miRNA-320b on THP-1-derived macrophage cholesterol efflux and AS by targeting on EEPD1（1）Population data.Bioinformatics analysis and microarray data were used to identify miRNAs targeting on EEPD1 gene.Firstly,Bioinformatics analysis was conducted to find miRNAs targeting on EEPD1,and then microarray was conducted to profile miRNA expression in peripheral blood mononuclear cells（PBMCs）of 24 CAD patients and seven healthy controls to identify differentially expressed miRNAs targeting on EEPD1,and qPCR was used to validate the differentially expressed miRNAs and the target genes in PBMCs of 123 CAD patients and 104 healthy controls.（2）Cellular research.Luciferase assay was conducted to evaluate the combination effect of miRNAs and EEPD1 gene,and then THP-1-derived macrophages were used to conduct functional studies.In order to verify whether the predicted EEPD1 and ABCG1were the target genes of miR-320b,luciferase assay was conducted to evaluate the activity the wild type and mutant reporter construct containing pmirGLO-EEPD1/3’-UTR and pmirGLO-ABCG1/3’-UTR,respectively.qPCR and WB were conducted to verify the effect of miR-320b on EEPD1,ABCG1 and ABCA1 in THP-1-derived macrophages.Fluorescence intensity was used to detect the effect of miR-320b on NBD-cholesterol efflux induced by high-density lipoprotein（HDL）and lipid-free apolipoprotein A1（apoAl）in THP-1-derived macrophage,respectively.（3）Animal models.Construction of adeno-associated virus（AAV）2-mediated macrophage-specific miR-320b overexpression mouse model to explore the effect of miR-320b on macrophage cholesterol efflux and AS in vivo.Apoe-/-mice were injected with AAV2-miR-320b to establish macrophage-specific miR-320b overexpression mouse model,thereafter fed with 14-week atherogenic diet to study the roles of miR-320b in vivo.Peritoneal macrophages were collected to detect HDL and apoAl induced NBD-cholesterol efflux.Oil red O staining was used to detect aorta lipid deposition and the cell composition of aortic plaque was detected by immunohistochemistry in paraffin section.2.The effect and molecular mechanism of EEPD1 gene on blood lipid metabolism.（1）Cellular research.Clarification of the molecular mechanism of EEPD1 on lipid metabolism-related genes in hepatocytes.Loss-of-function and gain-of-function strategies were conducted in this study.qPCR and WB were used to detect the mRNA and protein levels of lipid metabolism-related genes in HepG2 cells by overexpressing and knocking down of EEPD1 in HepG2 cells,respectively.To clarify the molecular mechanism of EEPD1 on regulating lipid metabolism genes,microarray assay was conducted to profile gene expression between EEPD1 knockdown HepG2 cells and the controls.Microarray data combined with Gene Ontology annotation（GO）analysis and Kyoto Encyclopedia of Genes and Genome（KEGG）pathway analysis was used to identify the differentially expressed genes in regulating lipid metabolism.The interaction between proteins of EEPD1 potential target genes was further detected by co-immunoprecipitation（CoIP）technology.（2）Animal models.Eepd1 liver-specific knockout and EEPD1 liver-specific overexpression mouse model were established to explore the effect of EEPD1on plasma lipid levels.C57BL/6 mice were injected with AAV8-EEPD1 via tail vein and fed with high-fat diet for 6 weeks to establish AAV8-mediated liver-specific EEPD1 overexpression hyperlipidemia mouse model.Additionally,Eepdl liver-specific knockout mice were established by CRISPR-Cas9 system,and fed with high-fat diet for 16 weeks.qPCR and WB were conducted to detect the mRNA and protein levels of lipid metabolism-related genes in liver,respectively.Cholesterol and Triglyceride determination kit was used to detect blood lipid levels,and oil red O staining was used to detect lipid deposition of liver frozen sections.Results1.The effect and molecular mechanism of miR-320b on THP-1-derived macrophage cholesterol efflux and AS by targeting on EEPD1.（1）Population data.miR-320b was increased while ABCG1 and EEPD1 genes were low expressed in CAD patients.Bioinformatics analysis indicated that ABCG1 and EEPD1 might be potential targets of miR-320b,microarray analysis showed that miR-320b was increased in CAD patients.Further qPCR analysis confirmed that miR-320b was highly expressed while ABCG1 and EEPD1 genes were low expressed in CAD patients compared with healthy controls.（2）Cellular research.ABCG1 and EEPD1 were the target genes of miR-320b,and participated in cholesterol efflux from macrophages.Dual-Luciferase Reporter Assay confirmed that ABCG1 and EEPD1 were the target genes of miR-320b,and miR-320b decreased HDL-and apoAl-mediated cholesterol efflux from macrophages partly by directly targeting on ABCG1/EEPD1 and partly via suppressing liver X receptors α（LXRα）-ABCA1/ABCG1 pathway.（3）Animal models.AAV2-mediated mouse macrophage-specific overexpression of miR-320b promoted the progress of AS.In vivo administration of AAV2-miR-320b into Apoe-/-mice showed the effective overexpression of miR-320b in peritoneal macrophages.Results showed attenuated cholesterol efflux from peritoneal macrophages with reduced expression of ABCA1,ABCG1 and EEPD1,and increased lipid LDL-c level with the down-regulation of hepatic LDLR while decreased lipid HDL-c level with the down-regulation of hepatic ABCA1.Meanwhile,AAV2-miR-320b treatment also enhanced pro-inflammatory cytokines levels,including Monocyte Chemoattractant Protein-1（MCP-1）、Interleukin-6（IL-6）and C-X-C Motif Chemokine Ligand 5（CXCL5）through the elevated phosphorylation level of nuclear factor-κB（NF-κB）p65 in macrophages.AAV2-miR-320b treatment increased atherosclerotic plaque size and lesional macrophage content and promoted the progress of AS.2.The effect and molecular mechanism of EEPD1 gene on blood lipid metabolism（1）Cellular research.EEPD1 regulated LDLR protein level by regulating the interaction among Cytochrome P450 Family 1 Subfamily B Member 1（CYP1B1）,proprotein convertase subtilisin/kexin type 9（PCSK9）and LDLR proteins.EEPD1 could regulate the protein level of PCSK9 and LDLR in HepG2 cells.Microarray data combined with GO and KEGG analysis showed that EEPD1 could modulate the expression of CYP1B1.qPCR and WB experiments verified that CYP1B1 was the target gene of EEPD1.CoIP experiment showed that EEPD1 could regulate PCSK9 protein level by ubiquitin modification of PCSK9 in the protein complex of CYP1B1,PCSK9 and LDLR,thus affected LDLR protein level.（2）Animal models.EEPD1 gene regulated mouse blood lipid levels.In vivo experiments showed that overexpression of EEPD1 in C57BL/6 mice reduced plasma LDL-c level with increased liver LDLR protein level,and increased liver lipid deposition with enhanced protein expression of LXRα and sterol regulatory element binding protein 1c（SREBP1c）in the liver,while reduced plasma TG level by inhibiting liver VLDL secretion.Eepd1 liver-specific knockout mice displayed increased plasma LDL-c level with the down-regulation of liver LDLR protein level,and attenuated liver lipid deposition with decreased protein expression of LXRa and SREBP1c in the liver,while increased plasma TG level by promoting liver VLDL secretion.Conclusion1)miR-320b decreased HDL-and apoAl-mediated cholesterol efflux from THP-1-derived macrophage partly by directly targeting ABCG1 and EEPD1 and partly via suppressing LXRα-ABCA1/ABCG1 pathway.2)In vivo administration of AAV2-miR-320b into Apoe-/-mice showed attenuated cholesterol efflux from peritoneal macrophages with reduced expression of ABCA1,ABCG1 and EEPD1,also increased lipid LDL-c level,and decreased lipid HDL-c level.Meanwhile,AAV2-miR-320b treatment also enhanced pro-inflammatory cytokines levels,increased atherosclerotic plaque size and lesional macrophage content and promoted the progress of AS.3)EEPD1 could modulate CYP1B1 expression at transcriptional level,and regulate PCSK9 protein level by ubiquitin modification of PCSK9 in the protein complex of CYP1B1,PCSK9 and LDLR to regulate the LDLR protein level.4)In vivo experiments showed that liver-specific overexpression of EEPD1 in C57BL/6 mice decreased PCSK9 protein level and increased LDLR protein level,thus reduced plasma LDL-c levels.AAV8-EEPD1 treatment also increased liver lipid deposition with enhanced protein expression of LXRa and SREBP1c in the liver while decreased plasma TG level by inhibiting liver VLDL secretion.Eepdl liver-specific knockout mice showed increased PCSK9 protein level and decreased LDLR protein level,thus displayed increased plasma LDL-c level.Eepd 1 liver-specific knockout mice also showed attenuated liver lipid deposition with decreased protein expression of LXRa and SREBPlc while increased plasma TG level by enhancing liver VLDL secretion.5)EEPD1 plays an important role in regulating lipid metabolism and AS,and might provide scientific evidence and potential intervention target for the prevention and treatment of CAD in the future. |
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