The Role And Mechanism Of Lipid Susceptibility Gene CCDC92 In Regulating LDL-c Metabolism

Background and Objective:Hyperlipidemia,characterized by blood elevated level of total cholesterol(TC)or low density lipoprotein-cholesterol(LDL-c),is one of the crucial independent risk factors for various cardiovascular diseases.Early and active control of plasma cholesterol concentration,especially of plasma TC and LDL-c level,is of great significance for the prevention and treatment of many cardiovascular diseases.CCDC92 gene was located in the susceptible loci of dyslipidemia and coronary heart disease(CHD)identified by genome-wide associate studies(GWASs).Genetic analysis showed that this gene might be related to the average particle size of blood cholesterol particles and the dysfunction of adipose tissue through insulin resistance,playing a potential role in the occurrence and development of coronary artery disease(CAD).In this study,we investigated the biological function of CCDC92 gene in LDL-c metabolism in vitro and in vivo through gain-and loss-of-function strategies.Furthermore,RNA sequencing was performed to profile the expression pattern of long non-coding RNA(lncRNA)and the regulatory network of competitive endogenous RNA(ceRNA)in hyperlipidemic mice model,from which crucial IncRNA-mediated ceRNA axises that might involve in the pathogenesis of dyslipidemia were further screen out.Methods:qRT-PCR and Western blot were performed to detect the expression changes of classic lipid metabolic genes,including LDLR,PCSK9,SREBP2 and IDOL,when CCDC92 gene was over expressed or knocked down in HepG2 cells.ELISA was used to detect the content of PCSK9 protein in the supernatant fluid of cultured HepG2 cells.Liver-targeted knockdown or overexpression of CCDC92 gene in C57BL/6J wild-type mice and ApoE-/-mice was successfully performed through tail intravenous injection with adeno-associated virus type 8(AAV8),Mice were fed with a chow diet or high-fat high-cholesterol(HFC)diet for different time points,and liver and plasma samples were collected for further investigation.qRT-PCR and Western blot were performed to detect the transcriptional and post-transcriptional expression level of classic lipid metabolic genes in mice liver,respectively.ELISA was used to detect the content of PCSK9 protein in mice plasma.Cholesterol analysis kits were used to detect the level of TC,LDL-c,HDL-c and TG in mice plasma.RNA sequencing was performed on six liver samples of ApoE-/-mice treated with AAV8-TBG-Ctrl or AAV8-TBG-CCDC92 and fed with HFC diet for 12 weeks.Cluster analysis was performed to identify the differentially expressed genes by FC>1.5 and P<0.05.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)analyses were performed on the differentially expressed genes to further explore the potential targets and mechanisms of CCDC92 gene in circulation cholesterol homeostasis.Hyperlipidemic mice model was constructed by feeding ApoE-/-mice with chow or HFC diet for 12 weeks.LncRNA/miRNA/mRNA sequencing was performed to identify the differentially expressed lncRNAs,miRNAs and mRNAs in the liver.GO and KEGG analyses were performed on the differentially expressed mRNAs to explore their potential functions.Bioinformatic analyses were used to analyze the connection between lncRNA-mRNA co-expression profile,miRNA-mRNA targeted profile,and lncRNA-miRNA co-expression and targeted profile.The lncRNA-medicated ceRNA network was constructed and crucial regulatory ceRNA axises were picked out based on the bioinformatic data and literature search.Results:In HepG2 cells,when CCDC92 gene was successfully knocked down through the transfection of siRNA,LDLR protein level in cells was significantly increased and PCSK9 protein level in cell supernatant fluid was significantly decreased.Instead,when CCDC92 gene was successfully over expressed through the infection of adenovirus,LDLR protein level in cells was significantly decreased and PCSK9 protein level in cell supernatant fluid was significantly increased.Through functional complementation assay,the knockdown of PCSK9 gene could partially reversed the downregulation of LDLR protein level induced by the overexpression of CCDC92 gene.C57BL/6J mice were injected with AAV8-TBG-CCDC92 or AAV8-TBG-control via tail vein,and fed with a chow diet or HFC diet for three weeks.Compared with the control mice,CCDC92 gene was successfully overexpressed in the liver of experimental mice.LDLR protein level in liver was significantly decreased,and PCSK9 protein level both in liver and in plasma were significantly increased.However,there was no significant change in liver mRNA level of classic lipid metabolic genes and plasma TC and LDL-c level.Similarly,C57BL/6J mice were injected with AAV8-Ccdc92-shRNA or AAV8-control-shRNA via tail vein,and fed with a chow diet or HFC diet for three weeks.Compared with the control mice,Ccdc92 gene was successfully knocked down in the liver of experimental mice.LDLR protein level in liver was significantly increased and PCSK9 protein level both in liver and in plasma were significantly decreased.However,there was no significant change in liver mRNA level of classic lipid metabolic genes and plasma TC and LDL-c level.Interestingly,when HFC-diet feeding time in C57BL/6J mice,which injected with AAV8-TBG-CCDC92 or AAV8-TBG-control via tail vein,prolonged to eighteen weeks,a significant up-regulated effect of plasma TC and LDL-c level could be seen in the experimental mice,compared with the control mice.ApoE-/-mice were injected with AAV8-TBG-CCDC92 or AAV8-TBG-control via tail vein,and fed with HFC diet for six or twelve weeks.The results of qRT-PCR,Western blot and ELISA were consistent with relative results in C57BL/6J mice fed with HFC diet for three weeks.Compared with the control mice,both TC and LDL-c level in plasma of experimental mice were significantly increased after a twelve-week HFC diet,rather than after a six-week HFC diet.Similarly,ApoE-/-mice were also injected with AAV8-Ccdc92-shRNA or AAV8-control-shRNA via tail vein,and fed with HFC diet for six or ten weeks.Compared with the control mice,both TC and LDL-c level in plasma of experimental mice were significantly decreased after a six-week HFC diet or a ten-week HFC diet.According to the transcriptome sequencing data,there were 1717 genes differentially expressed in the liver of ApoE-/-mice with overexpression of CCDC92 gene and a twelve-week HFC diet,including 840 up-regulated genes and 877 down-regulated genes.GO and KEGG analyses revealed that several genes and pathways,which involved in the cholesterol metabolism,were significantly changed.The differentially expressed genes were significantly enriched in pathways like cholesterol metabolism,fluid shear stress and atherosclerosis,fatty acid elongation,biosynthesis of unsaturated fatty acids,fat digestion and absorption,PPAR signaling pathway and ABC transport pathway etc.Furthermore,a total of 117 lncRNAs,53 miRNAs and 1689 mRNAs were identified to be differentially expressed in the mice liver with hyperlipidemia.GO analysis revealed that the differentially expressed mRNAs were significantly enriched in various biological processes including lipid metabolic process,fatty acid metabolic process and steroid metabolic process.KEGG analysis showed that the differentially expressed mRNAs were involved in the pathways like PPAR signaling pathway and biosynthesis of unsaturated fatty acids.The IncRNA-mediated ceRNA regulatory network for mice hyperlipidemic model were successfully constructed.Further analyses revealed that Inc-Dubr-mediated ceRNA axises might play an important role in the pathogenesis of dyslipidemia.Conclusion:In vitro experiments showed that in HepG2 cells,CCDC92 gene might regulate LDLR protein through modulating the secretion of PCSK9 protein in cell supernatant.In vivo experiments showed that in C57BL/6J mice fed with an eighteen-week HFC diet or ApoE-/-mice fed with a six-week or a twelve-week HFC diet,overexpression of CCDC92 gene caused upregulation of PCSK9 protein expression level in liver and plasma,downregulation of LDLR protein expression level in liver,and increase of both TC and LDL-c level in plasma of experimental mice.Instead,in ApoE-/-mice fed with a six-week or a ten-week HFC diet,knockdown of CCDC92 gene caused downregulation of PCSK9 protein expression level in liver and plasma,upregulation of LDLR protein expression level in liver,and decrease of both TC and LDL-c levels in plasma.Therefore,our study speculated that CCDC92 gene might regulate LDLR protein expression by modulating the expression and secretion of PCSK9 protein,and then play a function role in plasma LDL-c metabolism.Subsequently liver transcriptome sequencing showed that several genes and pathways proven to be important in the cholesterol metabolism were differentially expressed in mice injected with AAV8-TBG-CCDC92 and fed with a twelve-week HFC diet.CCDC92 gene is considered to become a potential target for the prevention and treatment of hyperlipidemia.Furthermore,the lncRNA-mediated regulatory network of ceRNA in mice liver with hyperlipidemia was successfully constructed,and the important ceRNA regulatory axises with potential biological function were further selected out.Our findings may provide new valuable markers and ideal targets for the development,progression and treatment of hyperlipidemia and other cardiovascular diseases.