乙型肝炎病毒X蛋白突变体致癌作用及其信号传导途径的研究
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摘要
乙型肝炎病毒(hepatitis B virus, HBV)与肝癌(HCC)的发生发展密切相关,乙肝病毒X开放读码框编码的X蛋白(HBx)作为反式激活因子在肝癌的发生中发挥重要的作用。但是,HBx致癌作用的分子机理仍不十分清楚。在肝癌组织中发现HBx基因普遍存在点突变和缺失突变,频率高达70%,但其作用不清。本研究为了进一步阐明HBx及其突变体在肝癌细胞中的作用和分子机制,从信号传导途径等方面探讨了野生型HBx促进肝癌细胞生长的分子机制；应用PCR技术从肝癌患者的癌组织和癌旁组织中筛选新的X基因突变体,并对已发现的HBx羧基端缺失突变体(命名为HBx△127)促进肝癌细胞生长的分子机制进行深入细致的研究。本论文从以下三个方面对HBx作用的分子机制进行了研究,主要研究内容如下：
第一部分：HBx促进肝癌细胞增殖信号传导途径研究
本研究探讨了5-脂氧合酶(5-lipoxygenases,5-LOX)在HBx促进肝癌细胞增殖中的作用。应用稳定表达HBx的人肝癌细胞系HepG2-X和H7402-X,通过real-time PCR、免疫印迹和酶联免疫分析等方法检测5-LOX表达水平,结果显示HBx能够上调5-LOX的表达,其细胞培养液中5-LOX代谢产物LTB4的含量增加；在此基础上,探讨HBx上调5-LOX表达的分子机制。结果显示,Nuclear factor-kappa B (NF-κB)的特异性抑制剂PDTC和NF-κB siRNA干扰片段可明显降低5-LOX的表达,表明HBx通过NF-κB上调了5-LOX的表达；然后,应用5-LOX的特异性抑制剂MK886和5-LOX siRNA处理HepG2-X (or H7402-X)细胞,发现5-LOX通过正反馈促进NF-κB的表达。因此,本研究结果提示,HBx通过5-LOX和NF-～B之间的正反馈调节环促进和维持肝癌细胞的生长。
第二部分：新的乙肝病毒X基因突变体的筛选
为了发现新的HBx基因突变,我们从60例肝癌病人的癌组织和癌旁组织中筛选HBx基因新的突变体。结果显示,44例癌组织和癌旁组织里均可检测到HBx基因,阳性率为73.3%；其中28例癌组织中HBx基因发生了突变(63.6%),27例的癌旁组织里HBx基因发生突变(61.3%)；测序结果显示,癌组织和癌旁组织中HBx点突变分别有35种不同类型,其中癌组织中HBx蛋白第30,33,38,144位氨基酸为热点突变位点,而第30,33和38位氨基酸伴随有组合突变(突变率30%),该突变体目前未见报道；癌旁组织中HBx蛋白的第31,40,87,94位氨基酸为热点突变位点,第94位氨基酸点突变(突变率11%)目前未见报道。在此基础上,对癌组织中发现的HBx组合突变体的功能进行了初步研究,通过报告基因检测了上述HBx组合突变体对NF-κB、hTERT、AP-1和survivin启动子转录活性的影响,结果显示第30,33和38位HBx的组合突变体与野生型的HBx蛋白结果相同,提示该组合突变并未影响其功能改变。
第三部分：HBx突变体HBx△127促进肝癌细胞增殖信号途径研究
我们实验室前期研究发现HBx△127与野生型HBx相比具有更强的促进肝癌细胞增殖的能力,但其分子机制尚不清楚。为了探讨HBx△127在肝癌发生、发展中的作用,本研究首先应用人肝癌细胞系HepG2和H7402,通过基因转染和G-418筛选等方法,获得了稳定转染HBx△127的肝癌细胞系,分别将其命名为HepG2-X△127和H7402-X△127;在此基础上,探讨了HBx△127对脂肪酸合成酶(Fatty acid synthase, FAS)和固醇调节元件结合蛋白-lc (Sterol regulatory element binding protein 1c, SREBP-1c)的影响。应用real-time PCR、荧光素酶报告基因、免疫印迹和酶联免疫分析等方法,发现HBx△127能够上调FAS和SREBP-1c的启动子活性及SREBP-1c的表达；进一步研究发现,5-LOX的特异性抑制剂MK886可明显抑制上述上调作用,提示HBx△127可通过5-LOX上调FAS的转录活性和SREBP-lc的表达,进而促进肝癌细胞的增殖；然后,我们进一步观察了HBx△127对5-LOX代谢产物LTB4的影响。应用酶联免疫吸附实验(ELISA)检测,发现HepG2-X△127和H7402-X△127细胞条件培养液中LTB4的含量与对照细胞组相比明显增加。当在培养液中外加100nM LTB4时,荧光素酶报告基因检测结果显示,FAS的启动子活性明显增强。最后,应用FAS的特异性抑制剂cerulenin处理HepG2-X△127和H7402-X△127细胞,5-LOX的表达受到抑制,提示FAS可调节5-LOX的表达。流式细胞术检测结果证实,FAS和5-LOX具有促进肝癌细胞增殖的作用。因此,本研究结果表明HBx△127具有促进和维持肝癌细胞生长的作用,与FAS和5-LOX之间的正反馈调节环有关。
综上所述,本研究为深入揭示HBx的致癌机理提供了新的实验依据,可进一步丰富HBx致癌的分子机理,对于肝癌的预防及治疗具有重要的理论价值。
Infection of hepatitis B virus (HBV) is closely related to the occurrence and development of hepatocellular carcinoma (HCC). HBV X protein (HBx), encoded by HBV X open reading frame (ORF), plays an important role in the development of HCC. Accordingly, it has been suggested that the multiply-sites mutation of HBx gene is universal and frequent (about 70%) in HCC tissue from patients. However, the mechanism for HBV-induced HCC remains unclear. In the present study, we examined the signal pathways of wild-type HBx protein to indicate the mechanism involving the promotion of heptoma cell growth mediated by HBx. HBx gene was amplified by HBx-specific PCR in tumor tissue samples and corresponding non-tumor tissue samples from Chinese patients with HCC to find sevel novel HBx mutants. We focused on investigating the mechanism involving the enhancement of heptoma cell growth mediated by the natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127). This study includes three parts as follows:
Part one:Investigation of the mechanism involving the promotion of cell growth mediated by HBx
We examined the effect of 5-LOX on promotion of cell growth mediated by HBx. The data showed that HBx was able to upregulate the expression of 5-LOX at levels of mRNA and protein. Enzyme-linked immunosorbent (ELISA) showed that the released leukotriene B4 (LTB4, a metabolite of 5-LOX) was increased in the conditioned media of human hepatoma HepG2-X and H7402-X cells. Moreover, we identified the mechanism involving the up-regulation of 5-LOX mediated by HBx. The data showed that the inhibition of NF-κB/p65 by PDTC (an inhibitor of NF-κB) or siRNA targeting NF-κB mRNA could attenuate the upregulation of 5-LOX mediated by HBx, suggesting that NF-κB was responsible for the upregulation of 5-LOX. Interestingly, our data showed that 5-LOX could activate NF-κB in a positive feedback manner by using MK886 (an inhibitor of 5-LOX) or siRNA targeting 5-LOX mRNA. Collectively, HBx promotes and keeps cell growth via a positive feedback involving 5-LOX and NF-κB.
Part two:Identification of HBx gene mutants in cancer and non-cancerous tissues from HCC patients
In this study, we try to find the novel HBx mutants in cancer and non-cancerous tissues from Chinese patients, and further investigate the characteristics of different type of HBx mutants. Therefore, HBx gene was amplified by HBx-specific PCR in 60 tumor tissue samples from Chinese patients with HCC and corresponding non-tumor tissue samples from the same group of HBsAg-positive patients. The data demonstrated that HBx gene was positive in 44 out of 60 tumor and non-tumor tissue samples (73.3%). We further analyzed the sequence of HBx gene, which showed that there were total 35 different types of point mutations in HBx protein from cancer tissues or non-cancerous tissues from 44 patients, respectively. Furthermore, we found that a novel phenotype of HBx gene multiple site mutation at the point of aa 30,33,38,88,144 from tumor samples and mutations at aa 31,40,87,94 from non-tumor samples were frequently detected. Importantly, the multiple site mutation at aa 30,33,38 from tumor samples and the single site mutation at aa 94 have never been reported by other study up to now.
Part three:Investigation of the mechanism involving the promotion of cell growth mediated by the mutant of HBx (HBx△127)
Previously, we identified a natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127), which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. First, we established the engineered cell lines HepG2-X△127/H7402-X△127 (stably transfected with the pCMV-X△127 plasmid). Then, we examined the effect of HBxA127 on the transcription of FAS and expression level of SREBP-lc. The data showed that HBxA127 strongly increased the promoter transcriptional activities of FAS and expression level of SREBP-lc by luciferase reporter gene assays, real-time PCR, ELISA and western blotting. Furthermore, we found that MK886 (an inhibitor of 5-LOX) could abolish the upregulation of FAS and SREBP-lc in a dose-dependent manner by luciferase reporter gene assays, real time RT-PCR and western blotting, suggesting that 5-LOX is responsible for the upregulation of FAS and SREBP-1c mediated by HBxA127. Moreover, we observed that HBx△127 could upregulate 5-LOX and resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX) in the conditioned media by enzyme-linked immunosorbent assay (ELISA). The additional LTB4 could upregulate the expression of FAS in the heptoma cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS). Collectively, HBx△127 promotes cell growth via a positive feedback loop involving 5-LOX and FAS, in which the released LTB4 is involved in the upregulation of FAS.
Taken together, our study provides new insight into the mechanism involving the promotion of cell growth mediated by HBx and the HBx mutant HBx△127. It is important for illustrating the molecular mechanism of HBX-related HCC, which is valuable in early diagnosis, prevention and therapy of HCC.
第一部分：HBx促进肝癌细胞增殖信号传导途径研究
本研究探讨了5-脂氧合酶(5-lipoxygenases,5-LOX)在HBx促进肝癌细胞增殖中的作用。应用稳定表达HBx的人肝癌细胞系HepG2-X和H7402-X,通过real-time PCR、免疫印迹和酶联免疫分析等方法检测5-LOX表达水平,结果显示HBx能够上调5-LOX的表达,其细胞培养液中5-LOX代谢产物LTB4的含量增加；在此基础上,探讨HBx上调5-LOX表达的分子机制。结果显示,Nuclear factor-kappa B (NF-κB)的特异性抑制剂PDTC和NF-κB siRNA干扰片段可明显降低5-LOX的表达,表明HBx通过NF-κB上调了5-LOX的表达；然后,应用5-LOX的特异性抑制剂MK886和5-LOX siRNA处理HepG2-X (or H7402-X)细胞,发现5-LOX通过正反馈促进NF-κB的表达。因此,本研究结果提示,HBx通过5-LOX和NF-～B之间的正反馈调节环促进和维持肝癌细胞的生长。
第二部分：新的乙肝病毒X基因突变体的筛选
为了发现新的HBx基因突变,我们从60例肝癌病人的癌组织和癌旁组织中筛选HBx基因新的突变体。结果显示,44例癌组织和癌旁组织里均可检测到HBx基因,阳性率为73.3%；其中28例癌组织中HBx基因发生了突变(63.6%),27例的癌旁组织里HBx基因发生突变(61.3%)；测序结果显示,癌组织和癌旁组织中HBx点突变分别有35种不同类型,其中癌组织中HBx蛋白第30,33,38,144位氨基酸为热点突变位点,而第30,33和38位氨基酸伴随有组合突变(突变率30%),该突变体目前未见报道；癌旁组织中HBx蛋白的第31,40,87,94位氨基酸为热点突变位点,第94位氨基酸点突变(突变率11%)目前未见报道。在此基础上,对癌组织中发现的HBx组合突变体的功能进行了初步研究,通过报告基因检测了上述HBx组合突变体对NF-κB、hTERT、AP-1和survivin启动子转录活性的影响,结果显示第30,33和38位HBx的组合突变体与野生型的HBx蛋白结果相同,提示该组合突变并未影响其功能改变。
第三部分：HBx突变体HBx△127促进肝癌细胞增殖信号途径研究
我们实验室前期研究发现HBx△127与野生型HBx相比具有更强的促进肝癌细胞增殖的能力,但其分子机制尚不清楚。为了探讨HBx△127在肝癌发生、发展中的作用,本研究首先应用人肝癌细胞系HepG2和H7402,通过基因转染和G-418筛选等方法,获得了稳定转染HBx△127的肝癌细胞系,分别将其命名为HepG2-X△127和H7402-X△127;在此基础上,探讨了HBx△127对脂肪酸合成酶(Fatty acid synthase, FAS)和固醇调节元件结合蛋白-lc (Sterol regulatory element binding protein 1c, SREBP-1c)的影响。应用real-time PCR、荧光素酶报告基因、免疫印迹和酶联免疫分析等方法,发现HBx△127能够上调FAS和SREBP-1c的启动子活性及SREBP-1c的表达；进一步研究发现,5-LOX的特异性抑制剂MK886可明显抑制上述上调作用,提示HBx△127可通过5-LOX上调FAS的转录活性和SREBP-lc的表达,进而促进肝癌细胞的增殖；然后,我们进一步观察了HBx△127对5-LOX代谢产物LTB4的影响。应用酶联免疫吸附实验(ELISA)检测,发现HepG2-X△127和H7402-X△127细胞条件培养液中LTB4的含量与对照细胞组相比明显增加。当在培养液中外加100nM LTB4时,荧光素酶报告基因检测结果显示,FAS的启动子活性明显增强。最后,应用FAS的特异性抑制剂cerulenin处理HepG2-X△127和H7402-X△127细胞,5-LOX的表达受到抑制,提示FAS可调节5-LOX的表达。流式细胞术检测结果证实,FAS和5-LOX具有促进肝癌细胞增殖的作用。因此,本研究结果表明HBx△127具有促进和维持肝癌细胞生长的作用,与FAS和5-LOX之间的正反馈调节环有关。
综上所述,本研究为深入揭示HBx的致癌机理提供了新的实验依据,可进一步丰富HBx致癌的分子机理,对于肝癌的预防及治疗具有重要的理论价值。
Infection of hepatitis B virus (HBV) is closely related to the occurrence and development of hepatocellular carcinoma (HCC). HBV X protein (HBx), encoded by HBV X open reading frame (ORF), plays an important role in the development of HCC. Accordingly, it has been suggested that the multiply-sites mutation of HBx gene is universal and frequent (about 70%) in HCC tissue from patients. However, the mechanism for HBV-induced HCC remains unclear. In the present study, we examined the signal pathways of wild-type HBx protein to indicate the mechanism involving the promotion of heptoma cell growth mediated by HBx. HBx gene was amplified by HBx-specific PCR in tumor tissue samples and corresponding non-tumor tissue samples from Chinese patients with HCC to find sevel novel HBx mutants. We focused on investigating the mechanism involving the enhancement of heptoma cell growth mediated by the natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127). This study includes three parts as follows:
Part one:Investigation of the mechanism involving the promotion of cell growth mediated by HBx
We examined the effect of 5-LOX on promotion of cell growth mediated by HBx. The data showed that HBx was able to upregulate the expression of 5-LOX at levels of mRNA and protein. Enzyme-linked immunosorbent (ELISA) showed that the released leukotriene B4 (LTB4, a metabolite of 5-LOX) was increased in the conditioned media of human hepatoma HepG2-X and H7402-X cells. Moreover, we identified the mechanism involving the up-regulation of 5-LOX mediated by HBx. The data showed that the inhibition of NF-κB/p65 by PDTC (an inhibitor of NF-κB) or siRNA targeting NF-κB mRNA could attenuate the upregulation of 5-LOX mediated by HBx, suggesting that NF-κB was responsible for the upregulation of 5-LOX. Interestingly, our data showed that 5-LOX could activate NF-κB in a positive feedback manner by using MK886 (an inhibitor of 5-LOX) or siRNA targeting 5-LOX mRNA. Collectively, HBx promotes and keeps cell growth via a positive feedback involving 5-LOX and NF-κB.
Part two:Identification of HBx gene mutants in cancer and non-cancerous tissues from HCC patients
In this study, we try to find the novel HBx mutants in cancer and non-cancerous tissues from Chinese patients, and further investigate the characteristics of different type of HBx mutants. Therefore, HBx gene was amplified by HBx-specific PCR in 60 tumor tissue samples from Chinese patients with HCC and corresponding non-tumor tissue samples from the same group of HBsAg-positive patients. The data demonstrated that HBx gene was positive in 44 out of 60 tumor and non-tumor tissue samples (73.3%). We further analyzed the sequence of HBx gene, which showed that there were total 35 different types of point mutations in HBx protein from cancer tissues or non-cancerous tissues from 44 patients, respectively. Furthermore, we found that a novel phenotype of HBx gene multiple site mutation at the point of aa 30,33,38,88,144 from tumor samples and mutations at aa 31,40,87,94 from non-tumor samples were frequently detected. Importantly, the multiple site mutation at aa 30,33,38 from tumor samples and the single site mutation at aa 94 have never been reported by other study up to now.
Part three:Investigation of the mechanism involving the promotion of cell growth mediated by the mutant of HBx (HBx△127)
Previously, we identified a natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127), which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. First, we established the engineered cell lines HepG2-X△127/H7402-X△127 (stably transfected with the pCMV-X△127 plasmid). Then, we examined the effect of HBxA127 on the transcription of FAS and expression level of SREBP-lc. The data showed that HBxA127 strongly increased the promoter transcriptional activities of FAS and expression level of SREBP-lc by luciferase reporter gene assays, real-time PCR, ELISA and western blotting. Furthermore, we found that MK886 (an inhibitor of 5-LOX) could abolish the upregulation of FAS and SREBP-lc in a dose-dependent manner by luciferase reporter gene assays, real time RT-PCR and western blotting, suggesting that 5-LOX is responsible for the upregulation of FAS and SREBP-1c mediated by HBxA127. Moreover, we observed that HBx△127 could upregulate 5-LOX and resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX) in the conditioned media by enzyme-linked immunosorbent assay (ELISA). The additional LTB4 could upregulate the expression of FAS in the heptoma cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS). Collectively, HBx△127 promotes cell growth via a positive feedback loop involving 5-LOX and FAS, in which the released LTB4 is involved in the upregulation of FAS.
Taken together, our study provides new insight into the mechanism involving the promotion of cell growth mediated by HBx and the HBx mutant HBx△127. It is important for illustrating the molecular mechanism of HBX-related HCC, which is valuable in early diagnosis, prevention and therapy of HCC.
引文
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