AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cell...AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.展开更多
Background:The liver plays a key role in regulating whole body cholesterol homeostasis.Hepatic cholesterol accumulation causes liver injury in fatty liver disease and hypercholesterolemia increases the risk of cardiov...Background:The liver plays a key role in regulating whole body cholesterol homeostasis.Hepatic cholesterol accumulation causes liver injury in fatty liver disease and hypercholesterolemia increases the risk of cardiovascular disease.MicroRNAs(miRNAs,miRs)have been shown to regulate various pathways in cholesterol metabolism.Recently,miR-185 has been shown to regulate sterol regulatory element-binding protein 2(SREBP2)and low-density lipoprotein receptor(LDLR)to modulate cholesterol syn-thesis and uptake.Materials and methods:The role of miR-185 in regulating diet-induced metabolic disorders were studied in liver-specific miRNA-185 knockout(L-miR-185 KO)mice.Results:L-miR-185 KO mice developed worsened hepatic steatosis upon high-fat high-cholesterol Western diet feeding with accumulation of triglyceride and cholesterol in the liver.In addition,L-miR-185 KO mice developed hypercholesterolemia upon Western diet feeding.Gene expression analysis showed that L-miR-185 KO mice did not show increased hepatic mRNA expression of SREBP2 or its targets LDLR and HMG-CoA reductase(HMGCR).Although expression of miR-185 mimic inhibited the mRNA of SREBP2,HMGCR and LDLR in HepG2 cells,miR-185 inhibitor did not increase the mRNA of SREBP2,HMGCR or LDLR in HepG2 cells.Conclusions:In conclusion,we reported that L-miR-185 KO mice were more sensitive to Western diet induced hepatic steatosis and hypercholesterolemia.The molecular mechanisms underlying these metabolic changes remain to be investigated in future studies.展开更多
基金Supported by Medical Specialty Development Projects of Beijing Municipal Administration of Hospitals,No.ZYLX201402Ministry of Education of The People’s Republic of China,No.20121107110012+1 种基金Beijing Municipal Commission of Education,No.11320016Collaborative Innovation Center of Infectious Diseases and Beijing Key Laboratory of Emerging Infectious Diseases,Beijing,China
文摘AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.
基金This study was supported by the USA National Institutes of Health(NIH)grant 1R01 DK117965-01A1(T.Li).
文摘Background:The liver plays a key role in regulating whole body cholesterol homeostasis.Hepatic cholesterol accumulation causes liver injury in fatty liver disease and hypercholesterolemia increases the risk of cardiovascular disease.MicroRNAs(miRNAs,miRs)have been shown to regulate various pathways in cholesterol metabolism.Recently,miR-185 has been shown to regulate sterol regulatory element-binding protein 2(SREBP2)and low-density lipoprotein receptor(LDLR)to modulate cholesterol syn-thesis and uptake.Materials and methods:The role of miR-185 in regulating diet-induced metabolic disorders were studied in liver-specific miRNA-185 knockout(L-miR-185 KO)mice.Results:L-miR-185 KO mice developed worsened hepatic steatosis upon high-fat high-cholesterol Western diet feeding with accumulation of triglyceride and cholesterol in the liver.In addition,L-miR-185 KO mice developed hypercholesterolemia upon Western diet feeding.Gene expression analysis showed that L-miR-185 KO mice did not show increased hepatic mRNA expression of SREBP2 or its targets LDLR and HMG-CoA reductase(HMGCR).Although expression of miR-185 mimic inhibited the mRNA of SREBP2,HMGCR and LDLR in HepG2 cells,miR-185 inhibitor did not increase the mRNA of SREBP2,HMGCR or LDLR in HepG2 cells.Conclusions:In conclusion,we reported that L-miR-185 KO mice were more sensitive to Western diet induced hepatic steatosis and hypercholesterolemia.The molecular mechanisms underlying these metabolic changes remain to be investigated in future studies.
