乙型肝炎病毒感染肝细胞癌患者中截短型中蛋白的研究
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摘要
目的:分子流行病学方法检测肝癌患者中HBV病毒株MHBst的种类,并研究各种MHBst的反式激活能力及对细胞的增殖、成瘤和凋亡影响。以免疫沉淀的方法筛选MHBst167候选结合蛋白。探讨截短中蛋白在肝细胞内的可能存在的分子作用机制。
方法:应用多聚酶链反应(PCR)和Alu-PCR技术自HBV感染肝癌患者外周血和肝癌组织中扩增S基因序列,并进行pMD18-T载体的TA克隆,随机选择部分克隆进行DNA测序并对测序结果进行生物信息学分析。应用GeneTailor Site-Directed Mutagenesis System突变S蛋白的起始密码子,将S基因点突变成功的不同MHBst构建真核表达载体和腺病毒表达载体,转染或感染细胞后Western-blot验证其蛋白的表达。通过启动子激活萤火虫萤光素酶的表达研究不同MHBst反式激活功能。通过CCK-8、软琼脂克隆形成及TUNEL方法观察不同截短型的中蛋白对细胞增殖、成瘤及凋亡功能的影响。以免疫沉淀的方法筛选出MHBst167结合蛋白。
结果:成功自80例HBV感染肝癌患者的血清中获得700多个S基因的阳性克隆,通过测序获得600条S区基因序列,发现7种分别在77,90,116,124,129,227,254处氨基酸终止编码的MHBst;其中129,227,254三种MHBst出现的频率较高,分别为15%(12/80),7.5%(6/80)和20%(16/80);12例MHBst129全为C基因型,6例MHBst227只有1例为B基因型,其余为C基因型,而16例254只有1例为C基因型,其余均为B基因型。Alu-PCR从30例肝癌标本中检测出3例标本有S基因的整合,共发现了9种整合位点。对8种不同MHBst和完整中蛋白成功进行了小蛋白起始密码子的突变。构建了8种不同MHBst和完整中蛋白的pCDNA3.1、p3xflag-cmv-10真核表达载体,并成功获得MOI值分别为:Ad-77:160,Ad-90:320,Ad-116:320,Ad-124:320,Ad-129:320,Ad-227:500,Ad-254:320,Ad-167:160,Ad-281:160,Ad-GFP:320的病毒液。获得了能够分别表达SV40、c-fos、c-myc启动子的细胞株。不同MHBst的反式激活能力、增殖及克隆形成能力不同。MHBst167可以通过TRAIL诱导产生细胞凋亡并可以被阻断剂PD98059。免疫沉淀方法获得了相互作用蛋白醛缩酶B。
结论:HBV感染肝癌患者血清中存在7种不同类型MHBst,以C基因型出现的种类比较多。HBV感染肝癌患者发生S基因整合较少,且为随机整合,但其整合可能涉及细胞的增殖、凋亡等生物学功能。不同类型的MHBst有反式激活能力,并对细胞的增殖及凋亡的有一定影响。免疫沉淀筛选出了醛缩酶B可与MHBst167蛋白相互作用,为下一步研究提供新的线索。
OBJECTIVE:To study the types of MHBst in hepatocellular carcinoma patiens and MHBst biologic funcation in hepatocytes.
METHODS:The S region of HBV was amplified by polymerase chain reaction (PCR) method from 80 cases of hepatocellular carcinoma, and the PCR products were cloned into pMD18-T vector and sequenced. Vector 10.0 software was emploied to analyze the sequences. Alu-polymerase chain reaction approach to detcet the HBV DNA integration into cellular genes. The PCR products were cloned into pMD18-T vector and sequenced. Apply NCBI Blast to analyze viral-host junctions. Artificial mutants used in this study were generated by GeneTailor Site-Directed Mutagenesis System. To express MHBst mutants in the mammalian cells, the MHBst fragment were cloned into the vector pcDNA3.1 by HindIII and BamH I sites. Positive clones were sequenced to confirm their compliance with the original results. Construction and expression of a recombinant adenovirus vector expressing MHBst. Construction the pGL3Basic-fos,myc-Luc reporter. SV40-Luc reporter from Promega reporter assay system. Apply CCK-8, soft agar assay, TUNEL methods to study MHBst biologic funcation. Immunoprecipitation to screen proteins interact with MHBst167.
RESULTS:After sequencing, obtained 600 sequence and find 7 types MHBst:77, 90, 116, 124, 129, 227, 254. Most 254 occur in B genotype and other types occur in C genotype. 129,227,254 types show higher frequencies,15%(12/80),7.5%(6/80)and 20%(16/80). Alu-PCR approach obtained 9 kinds of viral-host junctions. Successfully mutate S start codons. Constructed pCDNA3.1, p3×flag-cmv-10 and adenovirus AD-77, 90, 116, 124, 129, 227, 254,167,281 expression vector. Constructed SV40, c-fos、c-myc luciferase repoter expression vector and screen SV40,c-myc cell strain. MHBst show diffrenrnt transactive and clone form.MHBst can through TRAIL to induce cell apoptosis. MHBst show different trans-activation, proliferation and colony formation. MHBst167 interact with Aldolase by Immunoprecipitation.
CONCLUSION:There are 7 MHBst in HCC patients. Most MHBst belong to C genotyoe.HBV S gene show low integration rate in HCC and random integration,but the HBV S gene integration may involve in cell proliferation and apotoisis. This research indicates that HBV MHBst involve in trans-activation, cell proliferation and apoptosis. However, the roles of HBV envelope protein may be comprehensive and remain to be elucidated. MHBst167 interact with Aldolase offers an opportunity to probe into the role of MHBst in the pathogenesis of HBV-associated liver fibrosis and hepatic celluar carcinoma.
方法:应用多聚酶链反应(PCR)和Alu-PCR技术自HBV感染肝癌患者外周血和肝癌组织中扩增S基因序列,并进行pMD18-T载体的TA克隆,随机选择部分克隆进行DNA测序并对测序结果进行生物信息学分析。应用GeneTailor Site-Directed Mutagenesis System突变S蛋白的起始密码子,将S基因点突变成功的不同MHBst构建真核表达载体和腺病毒表达载体,转染或感染细胞后Western-blot验证其蛋白的表达。通过启动子激活萤火虫萤光素酶的表达研究不同MHBst反式激活功能。通过CCK-8、软琼脂克隆形成及TUNEL方法观察不同截短型的中蛋白对细胞增殖、成瘤及凋亡功能的影响。以免疫沉淀的方法筛选出MHBst167结合蛋白。
结果:成功自80例HBV感染肝癌患者的血清中获得700多个S基因的阳性克隆,通过测序获得600条S区基因序列,发现7种分别在77,90,116,124,129,227,254处氨基酸终止编码的MHBst;其中129,227,254三种MHBst出现的频率较高,分别为15%(12/80),7.5%(6/80)和20%(16/80);12例MHBst129全为C基因型,6例MHBst227只有1例为B基因型,其余为C基因型,而16例254只有1例为C基因型,其余均为B基因型。Alu-PCR从30例肝癌标本中检测出3例标本有S基因的整合,共发现了9种整合位点。对8种不同MHBst和完整中蛋白成功进行了小蛋白起始密码子的突变。构建了8种不同MHBst和完整中蛋白的pCDNA3.1、p3xflag-cmv-10真核表达载体,并成功获得MOI值分别为:Ad-77:160,Ad-90:320,Ad-116:320,Ad-124:320,Ad-129:320,Ad-227:500,Ad-254:320,Ad-167:160,Ad-281:160,Ad-GFP:320的病毒液。获得了能够分别表达SV40、c-fos、c-myc启动子的细胞株。不同MHBst的反式激活能力、增殖及克隆形成能力不同。MHBst167可以通过TRAIL诱导产生细胞凋亡并可以被阻断剂PD98059。免疫沉淀方法获得了相互作用蛋白醛缩酶B。
结论:HBV感染肝癌患者血清中存在7种不同类型MHBst,以C基因型出现的种类比较多。HBV感染肝癌患者发生S基因整合较少,且为随机整合,但其整合可能涉及细胞的增殖、凋亡等生物学功能。不同类型的MHBst有反式激活能力,并对细胞的增殖及凋亡的有一定影响。免疫沉淀筛选出了醛缩酶B可与MHBst167蛋白相互作用,为下一步研究提供新的线索。
OBJECTIVE:To study the types of MHBst in hepatocellular carcinoma patiens and MHBst biologic funcation in hepatocytes.
METHODS:The S region of HBV was amplified by polymerase chain reaction (PCR) method from 80 cases of hepatocellular carcinoma, and the PCR products were cloned into pMD18-T vector and sequenced. Vector 10.0 software was emploied to analyze the sequences. Alu-polymerase chain reaction approach to detcet the HBV DNA integration into cellular genes. The PCR products were cloned into pMD18-T vector and sequenced. Apply NCBI Blast to analyze viral-host junctions. Artificial mutants used in this study were generated by GeneTailor Site-Directed Mutagenesis System. To express MHBst mutants in the mammalian cells, the MHBst fragment were cloned into the vector pcDNA3.1 by HindIII and BamH I sites. Positive clones were sequenced to confirm their compliance with the original results. Construction and expression of a recombinant adenovirus vector expressing MHBst. Construction the pGL3Basic-fos,myc-Luc reporter. SV40-Luc reporter from Promega reporter assay system. Apply CCK-8, soft agar assay, TUNEL methods to study MHBst biologic funcation. Immunoprecipitation to screen proteins interact with MHBst167.
RESULTS:After sequencing, obtained 600 sequence and find 7 types MHBst:77, 90, 116, 124, 129, 227, 254. Most 254 occur in B genotype and other types occur in C genotype. 129,227,254 types show higher frequencies,15%(12/80),7.5%(6/80)and 20%(16/80). Alu-PCR approach obtained 9 kinds of viral-host junctions. Successfully mutate S start codons. Constructed pCDNA3.1, p3×flag-cmv-10 and adenovirus AD-77, 90, 116, 124, 129, 227, 254,167,281 expression vector. Constructed SV40, c-fos、c-myc luciferase repoter expression vector and screen SV40,c-myc cell strain. MHBst show diffrenrnt transactive and clone form.MHBst can through TRAIL to induce cell apoptosis. MHBst show different trans-activation, proliferation and colony formation. MHBst167 interact with Aldolase by Immunoprecipitation.
CONCLUSION:There are 7 MHBst in HCC patients. Most MHBst belong to C genotyoe.HBV S gene show low integration rate in HCC and random integration,but the HBV S gene integration may involve in cell proliferation and apotoisis. This research indicates that HBV MHBst involve in trans-activation, cell proliferation and apoptosis. However, the roles of HBV envelope protein may be comprehensive and remain to be elucidated. MHBst167 interact with Aldolase offers an opportunity to probe into the role of MHBst in the pathogenesis of HBV-associated liver fibrosis and hepatic celluar carcinoma.
引文
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1、Gish RG.Current treatment and future directions in the management of chronic hepatitis B viral infection. Clin Liver Dis. 2005 ;9(4):541-65 PMID: 16207563
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3、U Lauer, L Weiss, P H Hofschneider and A S Kekulé.The hepatitis B virus pre-S/S(t) transactivator is generated by 3' truncations within a defined region of the S gene. J Virol. 1992; 66(9): 5284-5289.PMID: 1323694
4、Ramesh R, Panda SK, Jameel S, Rajasambandam P.Mapping of the hepatitis B virus genome in hepatocellular carcinoma using PCR and demonstration of a potential trans-activator encoded by the frequently detected fragment.J Gen Virol. 1994;75:327-34 PMID: 8113754
5、Caselmann WH, Renner M, Schlüter V, Hofschneider PH, Koshy R, Meyer M.The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors.J Gen Virol. 1997;78:1487-95.PMID: 9191947
6、Hildt E, Munz B, Saher G, Reifenberg K, Hofschneider PH.The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice.EMBO J. 2002;21(4):525-35.PMID: 11847101
7、Liang X, Du J, Liu Y, Cui M, Ma C, Han L, Qu Z, Zhang Z, Sun Z, Zhang L, Chen YH, Sun W.The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2.Apoptosis. 2007;12(10):1827-36. PMID: 17701086
8、Hu YP, Yao YC, Li JX, Wang XM, Li H, Wang ZH, Lei ZH. The cloning of 3 truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice. World J Gastroenterol 2000;6:734-737. PMID: 11819684
9、Lauer U, Weiss L, Lipp M, Hofschneider PH, KekuléAS.The hepatitis B virus preS2/St transactivator utilizes AP-1 and other transcription factors for transactivation.Hepatology. 1994;19(1):23-31.PMID: 8276360
10、Hildt E, Hofschneider PH. The PreS2 activators of the hepatitis B virus:activators of tumour promotor pathways. Rec Res Cancer Res, 1998; 154: 315–329. PMID: 10027012
11、Liang X, Qu Z, Zhang Z, Du J, Liu Y, Cui M, Liu H, Gao L, Han L, Liu S, Cao L, Zhao P, Sun W.Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL.Biochem Biophys Res Commun. 2008;369(2):456-63.PMID: 18284918
12、Liu H, Luan F, Ju Y, Shen H, Gao L, Wang X, Liu S, Zhang L, Sun W, Ma C.In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase.Biochem Biophys Res Commun. 2007;355(2):379-84.PMID: 17307151
13、Luan F, Liu H, Gao L, Liu J, Sun Z, Ju Y, Hou N, Guo C, Liang X, Zhang L, Sun W, Ma C.Hepatitis B virus protein preS2 potentially promotes HCC development via its transcriptional activation of hTERT.Gut. 2009;58(11):1528-37.PMID: 19651630
14、Zoulim F, Locarnini S.Hepatitis B virus resistance to nucleos(t)ide analogues.Gastroenterology. 2009;137(5):1593-608.PMID: 19737565
15、Warner N, Locarnini S.The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound.Hepatology.2008;48(1):88-98. PMID: 18537180
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