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
目的：观察小鼠应激相关蛋白IFIT1（Interferon-induced protein with tetratricopetide repeats1）和JAB1（c-Jun平activation共处五项原则domain-binding protein 1）在肝脏的表达的细胞定位及氯胺酮对烧伤后早期肝脏IFIT1和JAB1表达...目的：观察小鼠应激相关蛋白IFIT1（Interferon-induced protein with tetratricopetide repeats1）和JAB1（c-Jun平activation共处五项原则domain-binding protein 1）在肝脏的表达的细胞定位及氯胺酮对烧伤后早期肝脏IFIT1和JAB1表达的影响，初步探讨氯胺酮调节炎症反应的分子机制。方法：将15只健康C57／129小鼠随机分为3组（n=5），正常对照组、烧伤组、烧伤＋氯胺酮组。采用TBSA 15～20％Ⅲ度小鼠烧伤模型；氯胺酮10mg／kg，肌肉注射，烧伤15min给予。分别于烧伤后4h脱臼处死，取肝。采用免疫印迹法（Western blot）察各组肝IFIT1和JAB1的表达情况。取正常对照组肝组织作病理切片，行免疫组织化学方法对IFIT1和JAB1的表达做细胞定位。结果：烧伤组IFIT1表达较正常对照组显著增高（P〈0．01）．烧伤组JAB1表达较正常对照组显著降低（P〈0．01）：氯胺酮治疗组IFIT1表达较正常对照组、烧伤对照组显著降低（P〈0．01）．氯胺酮治疗组JAB1表达较正常对照组显著降低（P〈0．05）、较烧伤组显著升高（P〈0．05）。免疫组织化学方法显示．IFIT1在肝细胞胞浆表达为阳性．JAB1在肝细胞胞浆和胞核均表达为阳性。结论：烧伤早期小鼠肝组织IFIT1表达增高而JJAB1表达降低，氯胺酮可降低烧伤早期小鼠肝组织IFIT1的高表达，增高JAB1表达；氯胺酮可通过调控严重烧伤应激中IFIT1和JAB1的表达而在全身炎性反应综合症（Systemic inflammatory response syndrome SIRS）中发挥作用。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.
文摘目的：观察小鼠应激相关蛋白IFIT1（Interferon-induced protein with tetratricopetide repeats1）和JAB1（c-Jun平activation共处五项原则domain-binding protein 1）在肝脏的表达的细胞定位及氯胺酮对烧伤后早期肝脏IFIT1和JAB1表达的影响，初步探讨氯胺酮调节炎症反应的分子机制。方法：将15只健康C57／129小鼠随机分为3组（n=5），正常对照组、烧伤组、烧伤＋氯胺酮组。采用TBSA 15～20％Ⅲ度小鼠烧伤模型；氯胺酮10mg／kg，肌肉注射，烧伤15min给予。分别于烧伤后4h脱臼处死，取肝。采用免疫印迹法（Western blot）察各组肝IFIT1和JAB1的表达情况。取正常对照组肝组织作病理切片，行免疫组织化学方法对IFIT1和JAB1的表达做细胞定位。结果：烧伤组IFIT1表达较正常对照组显著增高（P〈0．01）．烧伤组JAB1表达较正常对照组显著降低（P〈0．01）：氯胺酮治疗组IFIT1表达较正常对照组、烧伤对照组显著降低（P〈0．01）．氯胺酮治疗组JAB1表达较正常对照组显著降低（P〈0．05）、较烧伤组显著升高（P〈0．05）。免疫组织化学方法显示．IFIT1在肝细胞胞浆表达为阳性．JAB1在肝细胞胞浆和胞核均表达为阳性。结论：烧伤早期小鼠肝组织IFIT1表达增高而JJAB1表达降低，氯胺酮可降低烧伤早期小鼠肝组织IFIT1的高表达，增高JAB1表达；氯胺酮可通过调控严重烧伤应激中IFIT1和JAB1的表达而在全身炎性反应综合症（Systemic inflammatory response syndrome SIRS）中发挥作用。
