Id-1与HBx在乙肝相关性肝癌成瘤过程中相互作用的研究
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摘要
【背景】原发性肝癌(hepatocellular carcinoma,HCC)是全球发病率第五位的常见恶性肿瘤。在亚洲和大洋洲国家,特别是中国大陆,肝癌发病的主要诱因是慢性乙型肝炎病毒(hepatitis B,HBV)感染。对肝癌的早期诊断是提高肝癌患者疗效和预后的关键。目前包括肝癌切除术和肝移植在内的外科手术疗法仍是治疗早期肝癌的首选。然而,只有不到30%的患者能够在早期被诊断出肝癌并接受合适的治疗,大多数进展期患者由于治疗不及时而无法获得较理想的预后。由于我国众多的乙肝病毒携带者是肝癌的危险人群,更要求我们在早期发现并预测肝癌方面进行的研究,以期获得更好的肝癌的肿瘤标记物和预测因子。
乙肝病毒感染在肝癌的发病过程中起到了重要作用,在中国大陆,约80%以上的肝癌患者伴随着慢性乙肝病毒的感染。即便是HBV的隐匿性感染者,甚至是乙肝表面抗原(hepatitis B surface antigen,HBsAg)在体内已被廓清后的曾经感染者,其肝癌的发病风险仍高于普通人群。乙肝病毒可以整合入宿主基因组,并直接或间接的促进肝癌的发生。在这个过程中,乙肝病毒X蛋白(hepatitis B virus X protein,HBx protein)起到了重要的促进作用。HBx是由HBV-DNA中最短的开放读取框所编码的。它增强了HBV的转录,并激活了癌基因、细胞因子和生长因子等不同细胞的基因转录过程。HBx和其它多种因子如p53、DDB1、Caspase3和蛋白酶体亚单位等相互作用,促进细胞转化。在肝癌患者血清和肝癌组织中均可检测到高水平HBxAg和抗-HBx的表达。
Id蛋白即分化抑制蛋白或DNA结合抑制蛋白(Inhibitor of Differentiation and/or DNA-binding Protein)。由于Id蛋白缺少DNA结合结构域,它与其它碱性螺旋-环-螺旋转录因子(bHLH)结合后可形成没有DNA结合能力的异二聚体,起到了负性调节作用。作为Id蛋白家族的一员,Id-1在细胞分化过程中起到了重要的作用,它在包括肝癌在内的多种人类肿瘤中高表达。不受控制的Id-1蛋白表达将促进肿瘤细胞增殖、分化抑制、并刺激肿瘤血管生成及诱发基因组失稳。Id-1表达水平往往和肿瘤的高侵袭性、去分化程度和较差的临床预后相关。高表达的Id-1可以介导肝癌细胞的增殖,并且可成为预测慢性肝炎肝硬化患者肝癌发生风险的指标。Id-1可能在肝癌形成过程中的发挥重要作用,而它与HBV之间的关系值得进一步深入研究。
【目的】(1)检测Id-1和HBx蛋白在乙肝相关性肝癌组织中的表达情况。(2)分析乙肝相关性肝癌患者临床病理学特征与Id-1及HBx蛋白表达水平的相关性。(3)检测Id-1和HBx在肝癌细胞中的共定位情况。(4)检测HBx对肝癌细胞系中Id-1表达水平的影响。(5)构建人Id-1干扰慢病毒载体。(6)人Id-1干扰慢病毒载体对肝癌HepG2细胞的感染验证。
【方法】(1)对获得的96例乙肝相关性肝癌标本进行免疫组化染色,并对Id-1和HBx蛋白的染色结果进行分级。(2)借助SPSS软件,统计学分析Id-1和HBx的蛋白表达水平与肝癌临床病理学特征间的相关性以及Id-1的表达强度和乙肝相关性肝癌患者预后之间的关系。(3)用激光共聚焦免疫荧光染色法研究Id-1和HBx在肝癌细胞中的共定位情况。(4)Realtime-PCR和Western Blot分别检测Id-1 mRNA和蛋白在肝癌细胞系HepG2、HepG2.2.15、SMMC7721、FHCC98和肝细胞系HL7702中的表达情况。(5)Realtime-PCR和Western Blot检测Id-1蛋白在转染入HBx的肝癌HepG2-X和转染空质粒的HepG2-PC细胞内的表达情况。(6)采用荧光素酶报告基因系统检测Id-1启动子序列在HepG2-X和HepG2-PC细胞内的表达情况。(7)筛选获得效率最高的Id-1 RNA干扰序列并构建人Id-1干扰慢病毒载体。(8)验证人Id-1干扰慢病毒载体对HepG2细胞中Id-1的沉默效果。
【结果】(1)免疫组化染色发现,在96例乙肝相关性肝癌标本中,Id-1高表达率为64.6%,HBx的高表达率为74.0%。且Id-1的表达强度与HBx的表达强度正相关。(2)Id-1高表达水平与患者的血清高HBsAg水平、较差的肿瘤分化程度、门静脉侵犯、淋巴结转移及Child B/C级之间关系有统计学意义;Id-1高表达组的无瘤生存率和总生存率均较Id-1低表达组更差。(3)成对免疫组化染色和激光共聚焦免疫荧光染色发现Id-1和HBx在肝癌细胞中存在共定位现象,二者主要表达在细胞质和细胞核中。(4)经Realtime-PCR和Western Blot发现Id-1的mRNA和蛋白水平的表达强度在肝癌细胞系HepG2、HepG2.2.15、SMMC7721和FHCC98中均显著高于肝细胞系HL7702。(5)在肝癌HepG2-X中,Id-1的mRNA和蛋白表达强度均高于转染空质粒的HepG2-PC细胞。(6)报告基因检测发现在HepG2-X细胞中被激活的Id-1启动子的表达强度高于HepG2-PC细胞。(7)成功筛选并构建了人Id-1干扰慢病毒载体Id1-RNAi-LV,经鉴定病毒滴度为2.0×10E9 TU/mL;在HepG2细胞中,该载体对HepG2细胞中Id-1的mRNA和蛋白的表达均有明显抑制效果,抑制效率为61.2%。
【结论】(1)乙肝相关性肝癌标本中,Id-1和HBx均有表达,并且二者存在共定位现象。(2)Id-1蛋白的表达水平与乙肝相关性肝癌患者血清HBsAg水平、肿瘤分化程度、门静脉侵犯、淋巴结转移及Child分级等肿瘤恶性表型相关;Id-1高表达组患者有较差的预后。(3)Id-1在多种肝癌细胞系中均有表达,且其表达强度高于正常肝细胞系。(4)被转染入HepG2细胞中HBx,可提高Id-1启动子活性并使Id-1在mRNA及蛋白水平表达升高。(5)成功构建了Id-1干扰慢病毒载体Id1-RNAi-LV,并对肝癌HepG2细胞系中的Id-1基因有mRNA和蛋白水平的沉默效果。(6)综上Id-1可以作为乙肝相关性肝癌患者一个有用的预后指标。乙肝相关性肝癌标本中高表达的Id-1至少部分的受HBx表达所影响。构建成功的人Id-1干扰慢病毒载体可用于对Id-1参与肝癌机制的进一步研究,并可能成为新的治疗肝癌的分子靶向。
【Background】Hepatocellular carcinoma (HCC) is one of the most common malignant tumors world wide. In Asia-Pacific countries, especially in China, the main cause of HCC is chronic hepatitis B virus (HBV) infection. Early diagnosis is critical for better treatments and improved clinical outcomes. Patients with early-stage HCC can be treated using curative treatments, such as resection or liver transplantation. Nowadays, no more than 30% of HCC patients can be early diagnosed and received appropriate treatment. More patients with advanced HCC have much worse outcomes because of the delayed treatment. So, more studies focused on whether there are novel biomarkers that predict the risk of tumorigenesis and the survival time for large groups of HBV-infected patients are needed.
HBV plays an important role in HCC carcinogenesis. In China mainland, over 80% HCC patients are accompanied with chronic HBV infection. Moreover, the lifetime risk of HCC has been shown to be increased even in patients with occult HBV infection and after hepatitis B surface antigen (HBsAg) clearance. HBV could be integrated into the host genome to promote the carcinogenesis process by either directly or indirectly pathways. Hepatitis B virus X protein (HBx), one of the HBV-encoded proteins, accounts for the oncogenic properties of HBV in liver cells. In the pathogenesis of HBV-associated HCC cells, HBx may play a critical role by inducing progressive changes and facilitating the development of carcinoma. HBx could regulate different signaling pathways through interaction with a variety of proteins, such as the p53, DDB1, Caspase3 and proteasome subunit. HBxAg and anti-HBx are both over-expressed in the serum of HCC patients and tumor tissues.
Id proteins (Inhibitor of Differentiation and/or DNA-binding Protein) lack a basic domain and serve to inhibit the DNA binding of basic helix-loop-helix (bHLH) transcription factors by heterodimerization with bHLH proteins. As a member of the Id protein family, Id-1 plays a negative role in cell differentiation. The Id-1 gene has been shown to be highly expressed in a variety of primary human tumors including HCC. There is increasing evidence showing that deregulated Id-1 expression may contribute to various properties of tumor cells, including induction of aberrant cell proliferation, inhibition of differentiation, stimulation of angiogenesis and induction of genomic instability. Furthermore, the Id-1 expression level is often found to be correlated with malignant tumor phenotype, dedifferentiation and poor clinical outcomes of patients. Over-expressed Id-1 could induce proliferation of HCC cells and it may serve as a useful marker for determining HCC risk in patients with cirrhosis. So it is meaningful to further elucidate the role of Id-1 in HBV-related HCC.
【Objectives】(1) To detect the expression of Id-1 and HBx in HBV-related HCC tissue samples. (2) To analyzed the correlation between Id-1 and HBx expression levels and clinicopathological features of patients. (3) To detect whether Id-1 and HBx are co-localized in HCC cells. (4) To investigate the effect of HBx on Id-1 expression in HCC cell line. (5) To construct the lentiviral vector with Id-1 siRNA. (6) To measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells.
【Methods】(1) Tumor tissue samples obtained from a total of 96 HCC patients. The expression of Id-1 and HBx proteins of these samples are detected by immunohistochemical (IHC) staining and evaluated by two independent pathologists. (2) The corrections between the clinical pathological parameters and the IHC scores for Id-1 or HBx and the prognostic significance were statistical analysesed by SPSS software. (3) Immunofluorescent staining was used to detect the colocalization of Id-1 and HBx proteins in HCC cells. (4) The mRNA and protein expression of Id-1 in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) and hepatic cell line (HL7702) are detected and compared by Realtime-PCR and Western Blot. (5) The mRNA and protein expression of Id-1 in HepG2-X (stably transfected with HBx) and HepG2-PC (transfect with empty vector) are detected by Realtime-PCR and Western Blot. (6) Luciferase reporter gene vector with Id-1 promoter was used to investigate whether epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells. (7) Construct the lentiviral vector with Id-1 siRNA using the most effective Id-1 RNA interference sequence. (8) Measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells by Realtime-PCR and Western Blot.
【Results】(1) Over-expression of Id-1 and HBx were found in 64.6% and 74.0% of HBV-related HCC specimens, respectively. The expression of Id-1 was positively correlated to that of HBx. (2) Over-expression of Id-1 was correlated with the histological grade, portal vein invasion, lymph node metastasis, HBsAg and Child-Pugh classification. Patients with Id-1 overexpression had both shorter disease-free and overall survival times. (3) Colocalization of Id-1 and HBx was found by paired IHC and confocal study. They were both expressed in the cytoplasm and/or the nucleolus in HCC cells. (4) The expression of Id-1 mRNA and protein were both higher in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) than they were in hepatic cell line (HL7702) by Realtime-PCR and Western Blot. (5) HepG2-X showed a significantly higher Id-1 expression compared to HepG2-PC cells transfected with empty pcNDA3.1 plasmids, suggesting that HBx may up-regulate Id-1 in HCC cells as shown by real-time PCR and western blot. (6) Epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells by luciferase reporter gene system. (7) We successfully constructed the lentiviral vector with Id-1 siRNA named Id-1-RNAi-LV. The virus titer is 2.0×10E9 TU/mL. Id-1 mRNA and protein expression were significantly down-regulated after infected by Id-1-RNAi-LV in HepG2 cells. The silent ratio is 61.2%.
【Conclusion】(1) Id-1 overexpression was correlated with HBx expression, they were both colocolized in HCC cells of HBV-related HCC patients. (2) High expression of Id-1 were correlated with malignant HCC tumor phenotype such as serum HBsAg, histological grade, lymph node metastasis, portal vein invasion, Child-Pugh classification and poor clinical outcomes in HBV-related HCC. (3) Id-1 expression was higher in HCC cell lines than hepatic cell line. (4) Epitopic expression of HBx could stimulate Id-1 promoter. (5) Lentiviral vector with Id-1 siRNA was successfully constructed and have significantly silent efficiency in HepG2 cells. (6)Thus, Id-1 may serve as a useful prognosis marker for HBV-related HCC patients, and Id-1 overexpression in HBV-related HCC may be partially attributed to the effect of HBx. The lentiviral vector with Id-1 siRNA can be used for further researches on the mechanism of Id-1 involved in hepatocellular carcinoma and may act as a noval molecular targeting therapy of HBV-related HCC.
乙肝病毒感染在肝癌的发病过程中起到了重要作用,在中国大陆,约80%以上的肝癌患者伴随着慢性乙肝病毒的感染。即便是HBV的隐匿性感染者,甚至是乙肝表面抗原(hepatitis B surface antigen,HBsAg)在体内已被廓清后的曾经感染者,其肝癌的发病风险仍高于普通人群。乙肝病毒可以整合入宿主基因组,并直接或间接的促进肝癌的发生。在这个过程中,乙肝病毒X蛋白(hepatitis B virus X protein,HBx protein)起到了重要的促进作用。HBx是由HBV-DNA中最短的开放读取框所编码的。它增强了HBV的转录,并激活了癌基因、细胞因子和生长因子等不同细胞的基因转录过程。HBx和其它多种因子如p53、DDB1、Caspase3和蛋白酶体亚单位等相互作用,促进细胞转化。在肝癌患者血清和肝癌组织中均可检测到高水平HBxAg和抗-HBx的表达。
Id蛋白即分化抑制蛋白或DNA结合抑制蛋白(Inhibitor of Differentiation and/or DNA-binding Protein)。由于Id蛋白缺少DNA结合结构域,它与其它碱性螺旋-环-螺旋转录因子(bHLH)结合后可形成没有DNA结合能力的异二聚体,起到了负性调节作用。作为Id蛋白家族的一员,Id-1在细胞分化过程中起到了重要的作用,它在包括肝癌在内的多种人类肿瘤中高表达。不受控制的Id-1蛋白表达将促进肿瘤细胞增殖、分化抑制、并刺激肿瘤血管生成及诱发基因组失稳。Id-1表达水平往往和肿瘤的高侵袭性、去分化程度和较差的临床预后相关。高表达的Id-1可以介导肝癌细胞的增殖,并且可成为预测慢性肝炎肝硬化患者肝癌发生风险的指标。Id-1可能在肝癌形成过程中的发挥重要作用,而它与HBV之间的关系值得进一步深入研究。
【目的】(1)检测Id-1和HBx蛋白在乙肝相关性肝癌组织中的表达情况。(2)分析乙肝相关性肝癌患者临床病理学特征与Id-1及HBx蛋白表达水平的相关性。(3)检测Id-1和HBx在肝癌细胞中的共定位情况。(4)检测HBx对肝癌细胞系中Id-1表达水平的影响。(5)构建人Id-1干扰慢病毒载体。(6)人Id-1干扰慢病毒载体对肝癌HepG2细胞的感染验证。
【方法】(1)对获得的96例乙肝相关性肝癌标本进行免疫组化染色,并对Id-1和HBx蛋白的染色结果进行分级。(2)借助SPSS软件,统计学分析Id-1和HBx的蛋白表达水平与肝癌临床病理学特征间的相关性以及Id-1的表达强度和乙肝相关性肝癌患者预后之间的关系。(3)用激光共聚焦免疫荧光染色法研究Id-1和HBx在肝癌细胞中的共定位情况。(4)Realtime-PCR和Western Blot分别检测Id-1 mRNA和蛋白在肝癌细胞系HepG2、HepG2.2.15、SMMC7721、FHCC98和肝细胞系HL7702中的表达情况。(5)Realtime-PCR和Western Blot检测Id-1蛋白在转染入HBx的肝癌HepG2-X和转染空质粒的HepG2-PC细胞内的表达情况。(6)采用荧光素酶报告基因系统检测Id-1启动子序列在HepG2-X和HepG2-PC细胞内的表达情况。(7)筛选获得效率最高的Id-1 RNA干扰序列并构建人Id-1干扰慢病毒载体。(8)验证人Id-1干扰慢病毒载体对HepG2细胞中Id-1的沉默效果。
【结果】(1)免疫组化染色发现,在96例乙肝相关性肝癌标本中,Id-1高表达率为64.6%,HBx的高表达率为74.0%。且Id-1的表达强度与HBx的表达强度正相关。(2)Id-1高表达水平与患者的血清高HBsAg水平、较差的肿瘤分化程度、门静脉侵犯、淋巴结转移及Child B/C级之间关系有统计学意义;Id-1高表达组的无瘤生存率和总生存率均较Id-1低表达组更差。(3)成对免疫组化染色和激光共聚焦免疫荧光染色发现Id-1和HBx在肝癌细胞中存在共定位现象,二者主要表达在细胞质和细胞核中。(4)经Realtime-PCR和Western Blot发现Id-1的mRNA和蛋白水平的表达强度在肝癌细胞系HepG2、HepG2.2.15、SMMC7721和FHCC98中均显著高于肝细胞系HL7702。(5)在肝癌HepG2-X中,Id-1的mRNA和蛋白表达强度均高于转染空质粒的HepG2-PC细胞。(6)报告基因检测发现在HepG2-X细胞中被激活的Id-1启动子的表达强度高于HepG2-PC细胞。(7)成功筛选并构建了人Id-1干扰慢病毒载体Id1-RNAi-LV,经鉴定病毒滴度为2.0×10E9 TU/mL;在HepG2细胞中,该载体对HepG2细胞中Id-1的mRNA和蛋白的表达均有明显抑制效果,抑制效率为61.2%。
【结论】(1)乙肝相关性肝癌标本中,Id-1和HBx均有表达,并且二者存在共定位现象。(2)Id-1蛋白的表达水平与乙肝相关性肝癌患者血清HBsAg水平、肿瘤分化程度、门静脉侵犯、淋巴结转移及Child分级等肿瘤恶性表型相关;Id-1高表达组患者有较差的预后。(3)Id-1在多种肝癌细胞系中均有表达,且其表达强度高于正常肝细胞系。(4)被转染入HepG2细胞中HBx,可提高Id-1启动子活性并使Id-1在mRNA及蛋白水平表达升高。(5)成功构建了Id-1干扰慢病毒载体Id1-RNAi-LV,并对肝癌HepG2细胞系中的Id-1基因有mRNA和蛋白水平的沉默效果。(6)综上Id-1可以作为乙肝相关性肝癌患者一个有用的预后指标。乙肝相关性肝癌标本中高表达的Id-1至少部分的受HBx表达所影响。构建成功的人Id-1干扰慢病毒载体可用于对Id-1参与肝癌机制的进一步研究,并可能成为新的治疗肝癌的分子靶向。
【Background】Hepatocellular carcinoma (HCC) is one of the most common malignant tumors world wide. In Asia-Pacific countries, especially in China, the main cause of HCC is chronic hepatitis B virus (HBV) infection. Early diagnosis is critical for better treatments and improved clinical outcomes. Patients with early-stage HCC can be treated using curative treatments, such as resection or liver transplantation. Nowadays, no more than 30% of HCC patients can be early diagnosed and received appropriate treatment. More patients with advanced HCC have much worse outcomes because of the delayed treatment. So, more studies focused on whether there are novel biomarkers that predict the risk of tumorigenesis and the survival time for large groups of HBV-infected patients are needed.
HBV plays an important role in HCC carcinogenesis. In China mainland, over 80% HCC patients are accompanied with chronic HBV infection. Moreover, the lifetime risk of HCC has been shown to be increased even in patients with occult HBV infection and after hepatitis B surface antigen (HBsAg) clearance. HBV could be integrated into the host genome to promote the carcinogenesis process by either directly or indirectly pathways. Hepatitis B virus X protein (HBx), one of the HBV-encoded proteins, accounts for the oncogenic properties of HBV in liver cells. In the pathogenesis of HBV-associated HCC cells, HBx may play a critical role by inducing progressive changes and facilitating the development of carcinoma. HBx could regulate different signaling pathways through interaction with a variety of proteins, such as the p53, DDB1, Caspase3 and proteasome subunit. HBxAg and anti-HBx are both over-expressed in the serum of HCC patients and tumor tissues.
Id proteins (Inhibitor of Differentiation and/or DNA-binding Protein) lack a basic domain and serve to inhibit the DNA binding of basic helix-loop-helix (bHLH) transcription factors by heterodimerization with bHLH proteins. As a member of the Id protein family, Id-1 plays a negative role in cell differentiation. The Id-1 gene has been shown to be highly expressed in a variety of primary human tumors including HCC. There is increasing evidence showing that deregulated Id-1 expression may contribute to various properties of tumor cells, including induction of aberrant cell proliferation, inhibition of differentiation, stimulation of angiogenesis and induction of genomic instability. Furthermore, the Id-1 expression level is often found to be correlated with malignant tumor phenotype, dedifferentiation and poor clinical outcomes of patients. Over-expressed Id-1 could induce proliferation of HCC cells and it may serve as a useful marker for determining HCC risk in patients with cirrhosis. So it is meaningful to further elucidate the role of Id-1 in HBV-related HCC.
【Objectives】(1) To detect the expression of Id-1 and HBx in HBV-related HCC tissue samples. (2) To analyzed the correlation between Id-1 and HBx expression levels and clinicopathological features of patients. (3) To detect whether Id-1 and HBx are co-localized in HCC cells. (4) To investigate the effect of HBx on Id-1 expression in HCC cell line. (5) To construct the lentiviral vector with Id-1 siRNA. (6) To measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells.
【Methods】(1) Tumor tissue samples obtained from a total of 96 HCC patients. The expression of Id-1 and HBx proteins of these samples are detected by immunohistochemical (IHC) staining and evaluated by two independent pathologists. (2) The corrections between the clinical pathological parameters and the IHC scores for Id-1 or HBx and the prognostic significance were statistical analysesed by SPSS software. (3) Immunofluorescent staining was used to detect the colocalization of Id-1 and HBx proteins in HCC cells. (4) The mRNA and protein expression of Id-1 in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) and hepatic cell line (HL7702) are detected and compared by Realtime-PCR and Western Blot. (5) The mRNA and protein expression of Id-1 in HepG2-X (stably transfected with HBx) and HepG2-PC (transfect with empty vector) are detected by Realtime-PCR and Western Blot. (6) Luciferase reporter gene vector with Id-1 promoter was used to investigate whether epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells. (7) Construct the lentiviral vector with Id-1 siRNA using the most effective Id-1 RNA interference sequence. (8) Measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells by Realtime-PCR and Western Blot.
【Results】(1) Over-expression of Id-1 and HBx were found in 64.6% and 74.0% of HBV-related HCC specimens, respectively. The expression of Id-1 was positively correlated to that of HBx. (2) Over-expression of Id-1 was correlated with the histological grade, portal vein invasion, lymph node metastasis, HBsAg and Child-Pugh classification. Patients with Id-1 overexpression had both shorter disease-free and overall survival times. (3) Colocalization of Id-1 and HBx was found by paired IHC and confocal study. They were both expressed in the cytoplasm and/or the nucleolus in HCC cells. (4) The expression of Id-1 mRNA and protein were both higher in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) than they were in hepatic cell line (HL7702) by Realtime-PCR and Western Blot. (5) HepG2-X showed a significantly higher Id-1 expression compared to HepG2-PC cells transfected with empty pcNDA3.1 plasmids, suggesting that HBx may up-regulate Id-1 in HCC cells as shown by real-time PCR and western blot. (6) Epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells by luciferase reporter gene system. (7) We successfully constructed the lentiviral vector with Id-1 siRNA named Id-1-RNAi-LV. The virus titer is 2.0×10E9 TU/mL. Id-1 mRNA and protein expression were significantly down-regulated after infected by Id-1-RNAi-LV in HepG2 cells. The silent ratio is 61.2%.
【Conclusion】(1) Id-1 overexpression was correlated with HBx expression, they were both colocolized in HCC cells of HBV-related HCC patients. (2) High expression of Id-1 were correlated with malignant HCC tumor phenotype such as serum HBsAg, histological grade, lymph node metastasis, portal vein invasion, Child-Pugh classification and poor clinical outcomes in HBV-related HCC. (3) Id-1 expression was higher in HCC cell lines than hepatic cell line. (4) Epitopic expression of HBx could stimulate Id-1 promoter. (5) Lentiviral vector with Id-1 siRNA was successfully constructed and have significantly silent efficiency in HepG2 cells. (6)Thus, Id-1 may serve as a useful prognosis marker for HBV-related HCC patients, and Id-1 overexpression in HBV-related HCC may be partially attributed to the effect of HBx. The lentiviral vector with Id-1 siRNA can be used for further researches on the mechanism of Id-1 involved in hepatocellular carcinoma and may act as a noval molecular targeting therapy of HBV-related HCC.
引文
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