磷酸甘油酸激酶1启动子核心区域的确定及转录活性的分析
摘要
乙型肝炎病毒(hepatitis B virus, HBV)是嗜肝DNA病毒的一种,常导致严重肝病,包括肝硬化和肝细胞癌(hepatocellular carcinoma, HCC)。在HCC的形成过程中,乙型肝炎病毒(HBV)的慢性感染是引发肝细胞癌的主要危险因子。最近的研究发现,糖酵解与肿瘤的形成和预后关系密切。磷酸甘油酸激酶(Phosphoglycerate kinase PGK)是糖酵解的关键酶,在正常肝组织中,PGK1蛋白表达水平明显低于肝硬化和肝癌组织,HCC患者糖酵解作用增强,提示PGK1表达水平可作为HCC患者预后的独立指标。
目的：本研究旨在应用实时荧光PCR方法证实HBx蛋白对PGK1的反式激活作用；考察HBxAg对启动子活性的影响；验证核心启动子序列中SP1结合位点具有与肝癌相关细胞系的细胞核蛋白结合的活性。
方法：应用实时荧光定量PCR技术比较在HepG2.2.15细胞中与人HepG2细胞中PGK1在mRNA水平上的变化；构建PGK1-promoter荧光报告载体,通过分析其活性确定启动子的核心区域：应用凝胶迁移阻滞实验(EMSA)验证启动子核心区域的SP1位点与PGK1启动子结合活性,定点突变后检测其活性变化。
结果：本研究取得以下四方面的结果。首先,HepG2.2.15细胞中PGK1 mRNA的表达量是人肝癌HepG2细胞中的3.6倍,pcDNA3.1(-)-HBx转染HepG2细胞24小时后,经过实时荧光定量PCR检测,PGK1基因mRNA水平上调2.26倍。其次,实验中成功构建PGK1-promoter1-9系列荧光素报告质粒删除体,且测序证实插入序列与理论序列一致,并在HepG2细胞中得到表达。成功定位PGK1启动子的核心区域。第三方面,PGL4.10-PGK1-promoter 1分别与pcDNA3.1(-)-HBx及pcDNA3.1(-)共转染人肝癌HepG2细胞,试验组与对照组的转录活性相比提高1.86倍。第四方面,研究发现,SP1位点具有与PGK1启动子的结合活性,定点突变后其活性丧失。
结论：本研究的创新点表现在以下几个方面,HBV能够通过HBxAg上调PGK1基因表达水平；PGK1基因最小启动子活性区域位于转录起始位点上游152bp范围内(一247bp--95bp)；转录因子sp1参与PGK1转录调控过程。
本研究初步揭示了HBx蛋白能够反式激活PGK1基因的表达;HBx可提高PGK1启动子的转录活性以及SP1位点可能参与PGK1的转录调控；PGK1作为糖酵解相关蛋白在HBx致病机制中的作用,为PGK1成为HBV相关HCC患者预后指标提供了有价值的线索。
Hepatitis B virus (HBV) is one of the major pathogens of chronic hepatitis, which often leads to serious liver disease, including cirrhosis and hepatocellular carcinoma (HCC). In the formation of HCC, Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. Recent studies indicate that glycolysis is closly related to the formation of tumor and prognosis. Phosphoglycerate kinase (PGK) is the key enzyme in glycolysis. In the normal liver tissues, PGK1 protein level is significantly lower than that in cirrhosis and hepatocellular carcinoma, HCC patients with the enhanced role of glycolysis, suggesting that PGK1 expression level can be used as independent prognostic indicator for HCC patients.
Purpose:This study is to confirm HBx protein on the trans-activation of PGK1 by using real-time fluorescence PCR; to inspect the influence of HBxAg on promoter activity, and to check that whether SP1 binding sites in the core promoter can bind to the nuclear protein from hepatocellular related cells.
Method:We compare HepG2.2.15 cells and HepG2 cells at the mRNA level of PGK1 change using real-time fluorescence quantitative PCR technique. We constructed PGK1 fluorescent reporter vector and determined the active region by analyzing its activity, and choose gel mobility shift assay (EMSA) to verify the core promoter region of SP1 sites and the PGK1 promoter binding activity, and detect the site-directed mutagenesis changes.
Results:We obtained the results in the following four respects in this study. Firstly, HepG2.2.15 cells express 3.6-fold PGK1 mRNA than than that of human hepatoma HepG2 cells. Twenty-four hours after the transfection into the HepG2 cells with pcDNA3.1 (-)-HBx, measured by a real-time fluorescence quantitative PCR, the expression of PGK1 gene mRNA levels is found 2.26-fold increase. Secondly, PGK1-promoter 1-9 series of luciferase reporter plasmids truncated body is successfully constructed, and confirmed by DNA sequencing analysis that the insertion sequence consistent with the one theoretically expressed in HepG2 cells. We successfully targeted PGK1 core promoter region. Thirdly, PGL4.10-PGK1-promoter1 with pcDNA3.1 (-)-HBx, and pcDNA3.1 (-) respectively are co-transfected in human hepatoma HepG2 cells, and the transcriptional activity of the test group increased 1.86-fold are compared to the control group. Fourthly, the study have found that SP1 sites possessing the PGK1 promoter-binding activity and loss of its activity after site-directed mutagenesis.
conclusion:This study shows the following innovative aspects. HBV can up-regulate the PGK1 gene expression via HBxAg; PGK1 gene minimal promoter activity area is located at 152bp upstream of the transcription initiation site (-247bp--95bp); and Spl transcription factor involved in PGK1 transcription process.
This sdudy suggest that HBx protein trans-activate the PGK1 gene, HBx enhance the transcriptional activity of PGK1 promoter and SP1 sites be involved in the transcriptional regulation of PGK1, PGK1, as the glycolytic protein in the pathogenic mechanism of HBx provide a valuable information for PGK1 as a prognosis indicators of HBV-related HCC.
目的：本研究旨在应用实时荧光PCR方法证实HBx蛋白对PGK1的反式激活作用；考察HBxAg对启动子活性的影响；验证核心启动子序列中SP1结合位点具有与肝癌相关细胞系的细胞核蛋白结合的活性。
方法：应用实时荧光定量PCR技术比较在HepG2.2.15细胞中与人HepG2细胞中PGK1在mRNA水平上的变化；构建PGK1-promoter荧光报告载体,通过分析其活性确定启动子的核心区域：应用凝胶迁移阻滞实验(EMSA)验证启动子核心区域的SP1位点与PGK1启动子结合活性,定点突变后检测其活性变化。
结果：本研究取得以下四方面的结果。首先,HepG2.2.15细胞中PGK1 mRNA的表达量是人肝癌HepG2细胞中的3.6倍,pcDNA3.1(-)-HBx转染HepG2细胞24小时后,经过实时荧光定量PCR检测,PGK1基因mRNA水平上调2.26倍。其次,实验中成功构建PGK1-promoter1-9系列荧光素报告质粒删除体,且测序证实插入序列与理论序列一致,并在HepG2细胞中得到表达。成功定位PGK1启动子的核心区域。第三方面,PGL4.10-PGK1-promoter 1分别与pcDNA3.1(-)-HBx及pcDNA3.1(-)共转染人肝癌HepG2细胞,试验组与对照组的转录活性相比提高1.86倍。第四方面,研究发现,SP1位点具有与PGK1启动子的结合活性,定点突变后其活性丧失。
结论：本研究的创新点表现在以下几个方面,HBV能够通过HBxAg上调PGK1基因表达水平；PGK1基因最小启动子活性区域位于转录起始位点上游152bp范围内(一247bp--95bp)；转录因子sp1参与PGK1转录调控过程。
本研究初步揭示了HBx蛋白能够反式激活PGK1基因的表达;HBx可提高PGK1启动子的转录活性以及SP1位点可能参与PGK1的转录调控；PGK1作为糖酵解相关蛋白在HBx致病机制中的作用,为PGK1成为HBV相关HCC患者预后指标提供了有价值的线索。
Hepatitis B virus (HBV) is one of the major pathogens of chronic hepatitis, which often leads to serious liver disease, including cirrhosis and hepatocellular carcinoma (HCC). In the formation of HCC, Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. Recent studies indicate that glycolysis is closly related to the formation of tumor and prognosis. Phosphoglycerate kinase (PGK) is the key enzyme in glycolysis. In the normal liver tissues, PGK1 protein level is significantly lower than that in cirrhosis and hepatocellular carcinoma, HCC patients with the enhanced role of glycolysis, suggesting that PGK1 expression level can be used as independent prognostic indicator for HCC patients.
Purpose:This study is to confirm HBx protein on the trans-activation of PGK1 by using real-time fluorescence PCR; to inspect the influence of HBxAg on promoter activity, and to check that whether SP1 binding sites in the core promoter can bind to the nuclear protein from hepatocellular related cells.
Method:We compare HepG2.2.15 cells and HepG2 cells at the mRNA level of PGK1 change using real-time fluorescence quantitative PCR technique. We constructed PGK1 fluorescent reporter vector and determined the active region by analyzing its activity, and choose gel mobility shift assay (EMSA) to verify the core promoter region of SP1 sites and the PGK1 promoter binding activity, and detect the site-directed mutagenesis changes.
Results:We obtained the results in the following four respects in this study. Firstly, HepG2.2.15 cells express 3.6-fold PGK1 mRNA than than that of human hepatoma HepG2 cells. Twenty-four hours after the transfection into the HepG2 cells with pcDNA3.1 (-)-HBx, measured by a real-time fluorescence quantitative PCR, the expression of PGK1 gene mRNA levels is found 2.26-fold increase. Secondly, PGK1-promoter 1-9 series of luciferase reporter plasmids truncated body is successfully constructed, and confirmed by DNA sequencing analysis that the insertion sequence consistent with the one theoretically expressed in HepG2 cells. We successfully targeted PGK1 core promoter region. Thirdly, PGL4.10-PGK1-promoter1 with pcDNA3.1 (-)-HBx, and pcDNA3.1 (-) respectively are co-transfected in human hepatoma HepG2 cells, and the transcriptional activity of the test group increased 1.86-fold are compared to the control group. Fourthly, the study have found that SP1 sites possessing the PGK1 promoter-binding activity and loss of its activity after site-directed mutagenesis.
conclusion:This study shows the following innovative aspects. HBV can up-regulate the PGK1 gene expression via HBxAg; PGK1 gene minimal promoter activity area is located at 152bp upstream of the transcription initiation site (-247bp--95bp); and Spl transcription factor involved in PGK1 transcription process.
This sdudy suggest that HBx protein trans-activate the PGK1 gene, HBx enhance the transcriptional activity of PGK1 promoter and SP1 sites be involved in the transcriptional regulation of PGK1, PGK1, as the glycolytic protein in the pathogenic mechanism of HBx provide a valuable information for PGK1 as a prognosis indicators of HBV-related HCC.
引文
1. Hernandez M, Esteve T, Prat S, et al. Development of real time PCR systems based on SYBR R Green I, ArriplifluorTM and TaqMan R technologies for specific quantitative detection of the transgeriic maize event GA21. J Cereal Sci, 2004;39:99-107
2. Huang JF, Dai CY, Lin YY, et al. Performance characteristics of a real-time RT-PCR assay for quantification of hepatitis C virus RNA in patients with genotype 1 and 2 infections.Clin Chem Lab Med,2008;46:475-80
3. Lee SY, Choi MS, Lee D, et al. Overlapping gene mutations of hepatitis B virus in a chronic hepatitis B patient with hepatitis B surface antigen loss during lamivudine therapy. J Korean Med Sci,2005;20:433-437
4. Hsu CW, Yeh CT, Chang ML, et al. Identification of a hepatitis B virus S gene mutant in lamivudine-treated patients experiencing HBsAg seroclearance. Gastroenterology,2007; 132:543-550
5. Geng HF, Hua B, Wang H, et al. Dual-probe assay for detection of lamivudine-resistance hepatitis B virus by real-time PCR. J Virol Methods, 2006;132:25-31
6. Huang Y, Yang H, Borg BB, et al. A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection. Proc Natl Acad Sci USA,2007;104:85-90
7.李金明.实时荧光PCR技术.第一版.北京：人民军医出版社,2009；1-108,190-28
8.骆抗先.乙型肝炎基础和临床.第三版.北京：人民卫生出版社,20,06；245-260
9.施林祥,李东辉.实时荧光PCR研究新进展.世界华人消化杂志,2005；13(5)：596-599
10.任利,陈孝平,张万广等.乙肝病毒x蛋白激活NF-κB信号通路对AFP表达的影响.中国普通外科杂志,2008；17：768-772
11.王建军,杨倩,成军等.丙型肝炎病毒非结构蛋白HS5A反式激活基因HS5ATP6的克隆化研究.胃肠病学和肝病学杂志,2003；12(3)：251-253
12.徐志强,成军,张鸿飞等.应用表达谱芯片技术筛选HBcAg反式调节基因.世界华人消化杂志,2004;12(12)：2886-2890
13. Fu LY, Jia HL, Dong QZ, et al. Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses. BMC Cancer,2009；9:49
14.洪源,刘妍,成军等.应用表达谱芯片技术对截短型乙型肝炎病毒表面抗原中蛋白反式调节基因的研究.世界华人消化杂志,2003；11(7)：943-946
1. Katjana Daskalow,David Pfander, Wilko Weichert,et al.Distinct temporospatial expression patterns of glycolysis-related proteins in human hepatocellular carcinoma. Histochem Cell Biol 2009,(132):21-31.
2.德,吴忠道,余新炳.磷酸甘油酸激酶的研究进展.中国热带医学杂志,2005,5(2)：385-387
3.成军,李莉.清除乙型肝炎病毒的非细胞裂解机制.世界华人消化杂志2002,(10)：73-76
4.成军.肿瘤相关基因.北京：第1版.北京医科大学出版社,2000：1
5.成军,杨守纯.现代肝炎病毒分子生物学.北京：第1版.人民军医出版社,1997：20-25
6.成军.慢性病毒性肝炎发病机制的分子生物学研究.世界华人消化杂志.2002,(10)：125-128
7.刘妍,董菁,成军,钟彦伟,夏小兵,李克,王琳,施双双,段惠娟.乙型肝炎病毒x蛋白反式激活SV40毒早期启动子的研究.解放军医学杂志.2001,(26)：404-406
8.陆荫英,李克,成军,王琳,刘妍,段惠娟,张玲霞.乙型肝炎病毒X基因酵母表达载体构建及表达.世界华人消化杂志.2002,(10)：15-18
9.董菁,施双双,皇甫竞坤,成军,王勤环,李莉,斯崇文.乙型肝炎病毒x基因准种特点的研究.中国毒学.2002,(17)：22-26
10. Siddiqui A, Jameel S, Mapoles J. Expression of the hepatitis B virus X gene in mammalian cells.Proc Natl Acad Sci USA 1987,(84):2513-2517
11. Avantaggiati ML, Natoli G, Balsano C, Chirillo P, Artini M,De Marzio E, Collepardo D, Levrero M.The hepatitis B virus (HBV) pX transactivates the c-fos promoter through multiple cis-acting elements. Oncogene 1993,(8):1567-1574
12. Weil R, Sirma H, Giannini C, Kremsdorf D, Bessia C,Dargemont C, Brechot C, Israel A. Direct association and nuclear import of the hepatitis B virus X protein with the NFkappa B inhibitor Ikappa B alpha. Mol Cell Biol 1999;19:
13.成军.乙型肝炎病毒基因组结构与功能复杂性的研究进展1249 6345-6354
14. Su F, Theodosis CN, Schneider RJ. Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. J Virol 2001,(75):215-225
15. Tang H,Delgemma L.Huang FJ,et al.The transcriptional transactivation function of HBx protein is important for ita augmentation role in heoatitis B virus replication.J Virol,2005,79(9):5548-5556.
16. Murakami S.Hepatitis B virus X protein:a multifunctional viral regulator. J Gastroenterol 2001,36(10):651-660.
17. Kumar V, Jayasuryan N. Kumar R. A truncated mutant(residues 58-140) of the hepatitis B virus X protein retains transactivation function.Proc Natl Acad Sci USA,1996,93(11),5647-5652.
18. Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. Int J Cancer,2007, 15(21):1257-1264.
19. Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett,2007,18;252(2):157-170.
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21. Edmead C,Kanthou C,Benzakour O.Thrombin activates transcription factor SP1,NF-kappa B and CREB:Importance of the use of phosphatase inhibitors during nuclear protein extraction for the assesment of transcription factor DNA-binging activities[J],Anal Biochem,1999,275(2):180-186
22. Strowski M Z, Cramer T, Schafer G,et al. Helicobacter polyri stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK/ERK-dependent activation of SP1 and SP3[J].FASEB J,2004,18(1):218-220
23. Berg J M. Spl and the subfamily of zinc finger proteinswith guaninerich binding sites[J]. Proc Natl Acad Sci U S A,1992,89(23):11109-11110
24. Safe S, AbdelrahimM. SP transcription factor family and its role in cancer. Eur J Cancer,2005,41(16):2438-2448
25. Wang L,Wei D,HuangS,et al.Transcription factor SP1 expression is a significant predictor of survival in human gastric cancer[J].Clin Cancer Res,2003,9(17): 6371-6380
26. Murakami S.Hepatitis B virus X protein:structure,function andbiology. Inter-virology.1999,42(2-3):81-99
27. Barak O, Aronheim A,Shaul Y,HBV X protein targets HIV Tat-binding proteinl. Virology.2001.283(1):110-120
2. Huang JF, Dai CY, Lin YY, et al. Performance characteristics of a real-time RT-PCR assay for quantification of hepatitis C virus RNA in patients with genotype 1 and 2 infections.Clin Chem Lab Med,2008;46:475-80
3. Lee SY, Choi MS, Lee D, et al. Overlapping gene mutations of hepatitis B virus in a chronic hepatitis B patient with hepatitis B surface antigen loss during lamivudine therapy. J Korean Med Sci,2005;20:433-437
4. Hsu CW, Yeh CT, Chang ML, et al. Identification of a hepatitis B virus S gene mutant in lamivudine-treated patients experiencing HBsAg seroclearance. Gastroenterology,2007; 132:543-550
5. Geng HF, Hua B, Wang H, et al. Dual-probe assay for detection of lamivudine-resistance hepatitis B virus by real-time PCR. J Virol Methods, 2006;132:25-31
6. Huang Y, Yang H, Borg BB, et al. A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection. Proc Natl Acad Sci USA,2007;104:85-90
7.李金明.实时荧光PCR技术.第一版.北京：人民军医出版社,2009；1-108,190-28
8.骆抗先.乙型肝炎基础和临床.第三版.北京：人民卫生出版社,20,06；245-260
9.施林祥,李东辉.实时荧光PCR研究新进展.世界华人消化杂志,2005；13(5)：596-599
10.任利,陈孝平,张万广等.乙肝病毒x蛋白激活NF-κB信号通路对AFP表达的影响.中国普通外科杂志,2008；17：768-772
11.王建军,杨倩,成军等.丙型肝炎病毒非结构蛋白HS5A反式激活基因HS5ATP6的克隆化研究.胃肠病学和肝病学杂志,2003；12(3)：251-253
12.徐志强,成军,张鸿飞等.应用表达谱芯片技术筛选HBcAg反式调节基因.世界华人消化杂志,2004;12(12)：2886-2890
13. Fu LY, Jia HL, Dong QZ, et al. Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses. BMC Cancer,2009；9:49
14.洪源,刘妍,成军等.应用表达谱芯片技术对截短型乙型肝炎病毒表面抗原中蛋白反式调节基因的研究.世界华人消化杂志,2003；11(7)：943-946
1. Katjana Daskalow,David Pfander, Wilko Weichert,et al.Distinct temporospatial expression patterns of glycolysis-related proteins in human hepatocellular carcinoma. Histochem Cell Biol 2009,(132):21-31.
2.德,吴忠道,余新炳.磷酸甘油酸激酶的研究进展.中国热带医学杂志,2005,5(2)：385-387
3.成军,李莉.清除乙型肝炎病毒的非细胞裂解机制.世界华人消化杂志2002,(10)：73-76
4.成军.肿瘤相关基因.北京：第1版.北京医科大学出版社,2000：1
5.成军,杨守纯.现代肝炎病毒分子生物学.北京：第1版.人民军医出版社,1997：20-25
6.成军.慢性病毒性肝炎发病机制的分子生物学研究.世界华人消化杂志.2002,(10)：125-128
7.刘妍,董菁,成军,钟彦伟,夏小兵,李克,王琳,施双双,段惠娟.乙型肝炎病毒x蛋白反式激活SV40毒早期启动子的研究.解放军医学杂志.2001,(26)：404-406
8.陆荫英,李克,成军,王琳,刘妍,段惠娟,张玲霞.乙型肝炎病毒X基因酵母表达载体构建及表达.世界华人消化杂志.2002,(10)：15-18
9.董菁,施双双,皇甫竞坤,成军,王勤环,李莉,斯崇文.乙型肝炎病毒x基因准种特点的研究.中国毒学.2002,(17)：22-26
10. Siddiqui A, Jameel S, Mapoles J. Expression of the hepatitis B virus X gene in mammalian cells.Proc Natl Acad Sci USA 1987,(84):2513-2517
11. Avantaggiati ML, Natoli G, Balsano C, Chirillo P, Artini M,De Marzio E, Collepardo D, Levrero M.The hepatitis B virus (HBV) pX transactivates the c-fos promoter through multiple cis-acting elements. Oncogene 1993,(8):1567-1574
12. Weil R, Sirma H, Giannini C, Kremsdorf D, Bessia C,Dargemont C, Brechot C, Israel A. Direct association and nuclear import of the hepatitis B virus X protein with the NFkappa B inhibitor Ikappa B alpha. Mol Cell Biol 1999;19:
13.成军.乙型肝炎病毒基因组结构与功能复杂性的研究进展1249 6345-6354
14. Su F, Theodosis CN, Schneider RJ. Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. J Virol 2001,(75):215-225
15. Tang H,Delgemma L.Huang FJ,et al.The transcriptional transactivation function of HBx protein is important for ita augmentation role in heoatitis B virus replication.J Virol,2005,79(9):5548-5556.
16. Murakami S.Hepatitis B virus X protein:a multifunctional viral regulator. J Gastroenterol 2001,36(10):651-660.
17. Kumar V, Jayasuryan N. Kumar R. A truncated mutant(residues 58-140) of the hepatitis B virus X protein retains transactivation function.Proc Natl Acad Sci USA,1996,93(11),5647-5652.
18. Su PF, Lee TC, Lin PJ, et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. Int J Cancer,2007, 15(21):1257-1264.
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