焦童1,2, 刘宁宁1, 叶昕1
1.中国科学院微生物研究所, 北京 100101;
2.中国科学院大学, 北京 100049
收稿日期：2016-12-02；修回日期：2017-02-09；网络出版日期：2017-03-02
基金项目：国家重点基础研究发展计划（2015CB910502）；国家自然科学青年基金（31600129）
*通信作者：叶昕, Tel/Fax: +86-10-64807508; E-mail: yex@im.ac.cn
摘要：[目的]在细胞水平上研究乙型肝炎病毒X蛋白（HBx）对胰岛素样生长因子结合蛋白3（IGFBP3）转录的影响并对具体机制进行初步探索。[方法]首先采用RNA-Deep-Sequencing技术分析HepG2和乙型肝炎病毒（HBV）转基因细胞HepG2-4D14中表达差异的基因，然后通过实时定量PCR对相关基因进行验证；利用启动子报告基因分析，研究HBx对相关基因IGFBP3转录的调控；通过染色质免疫共沉淀方法，分析HBx抑制IGFBP3启动子活性的机制。[结果]RNA-Deep-Sequencing的结果表明IGFBP3在HBV转基因细胞HepG2-4D14中水平显著下调，实时定量PCR结果与RNA-Deep-Sequencing一致。进一步研究表明HBx能明显抑制IGFBP3的转录，通过实时定量PCR发现HBx对IGFBP3转录的抑制作用依赖于p53；染色质免疫共沉淀实验结果表明HBx能够通过抑制p53与IGFBP3启动子的结合，从而抑制IGFBP3的转录。IGFBP3是一种细胞周期负调控蛋白，我们推测HBx对IGFBP3水平的下调是其促进细胞增殖的途径之一。[结论]HBV能显著下调IGFBP3的转录，机制研究揭示HBV HBx通过干扰p53与IGFBP3启动子的结合进而抑制IGFBP3的转录。
关键词： HBx IGFBP3 p53
Mechanism of the inhibitory effect of HBx on IGFBP3 transcription
Jiao Tong1,2, Liu Ningning1, Ye Xin1
1.Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
2.University of Chinese Academy of Science, Beijing 100049, China
Received 2 December 2016; Revised 9 February 2017; Published online 2 March 2017
*Corresponding author: Xin Ye, Tel/Fax: +86-10-64807508; E-mail: yex@im.ac.cn
Supported by the Key Project of Chinese National Programs of Fundamental Research and Development (2015CB910502) and by the National Natural Science Foundation of China (31600129)
Abstract: [Objective]To study the mechanism of HBx regulating the transcription of insulin growth factor binding protein 3 (IGFBP3).[Methods]RNA-sequencing method was used to screen differently expressing genes in HepG2 and HBV transgenic cell line HepG2-4D14. The mRNA of IGFBP3 was measured by reverse transcription and real-time PCR. To verify the activity of IGFBP3 promoter, cells were analyzed by luciferase assay. The binding of p53 and IGFBP3 promoter was measured by ChIP Assay.[Results]The level of IGFBP3 mRNA in HBV transgenic cell line HepG2-4D14 was significantly lower than that in HepG2 cells. The data of real-time PCR indicated that HBV HBx can down regulate the transcription of IGFBP3. By taking the approach of promoter luciferase assay on HCT116 and HCT116-p53-/- cell lines, we found that HBx can inhibit the promoter activity of IGFBP3 in a p53-dependent manner. Our data also showed that_HBx can significantly interfere with the binding of p53 to the promoter of IGFBP3. As IGFBP3 is a suppressor for cell growth, we postulate that HBx promotes the cell proliferation by reducing the level of IGFBP3.[Conclusion]HBx can inhibit the transcription of IGFBP3 in a p53-dependent manner.
Key words: HBx IGFBP3 p53
原发性肝癌(HCC)是全球最普遍的恶性肿瘤之一，世界范围内有超过3.5亿慢性感染患者存在发展为原发性肝癌的风险，每年有60万人死于乙型肝炎病毒(HBV)的慢性感染[1]。HBV的慢性感染是导致HCC的最主要原因之一，目前对其具体机制的研究仍不清楚。
HBV属于嗜肝DNA病毒科，通过逆转录进行基因组复制。HBV基因组是长为3.2 kb的环状部分双链DNA，具有4个重叠的开放阅读框(ORFs)，分别编码病毒被膜蛋白(pre-S1/pre-S2/S)、核心蛋白(pre-C/C)、聚合酶(Pol)以及X蛋白(HBx)[2]。其中，HBx由154个氨基酸组成，分子量大小为17 KD，在所有哺乳动物嗜肝病毒中保守[3]。已有报道证实HBx在基因转录[3]、信号转导[4]、增殖凋亡[5]以及周期调控等方面具有重要的调节作用，并在HCC形成和发展的多个阶段发挥功能，比如，HBx能够抑制肿瘤抑制因子p53的转录及其与靶基因启动子的结合，从而发挥抗凋亡的作用[6-8]。
我们主要研究了HBV在原发性肝癌发生发展过程中所发挥的作用，以及HBV的慢性感染导致HCC的机制。为此我们采用RNA-Deep-Sequencing技术分析了HepG2和HBV转基因细胞HepG2-4D14中表达差异的基因，筛选其中能够影响细胞增殖、细胞凋亡以及肿瘤形成的基因，旨在阐明HBV通过调控基因表达导致HCC发生发展的机制。
胰岛素样生长因子结合蛋白(IGFBPs)家族由IGFBP1、IGFBP2、IGFBP3、IGFBP4、IGFBP5和IGFBP6组成。这些蛋白具有36%的同源性[9-10]，都能够结合胰岛素样生长因子(IGF)。IGFBP由3个结构域组成，N端及C端结构域与IGF的结合相关，在所有的IGFBP中保守。中间结构域存在差异，可能与不依赖IGF的功能有关[11]，这使得IGFBP能够通过IGF依赖性及非依赖性的机制发挥生物学功能。
IGFBP3是IGFBP家族中含量最高的成员，主要由肝脏产生，基因定位于7pl2-14，由264个氨基酸组成。IGFBP3可以与IGF结合形成复合物，延长IGF的半衰期，降低IGF与其受体的结合，从而竞争性地阻断IGFⅠ型受体介导的信号传导，间接抑制肿瘤生长[12]。
已有研究表明，p53能够与IGFBP3的启动子区结合，促进IGFBP3的转录。由此我们推测，HBx可能会通过p53抑制IGFBP3转录，进而解除IGFBP3的促凋亡作用。为了研究p53是否介导HBx对IGFBP3的抑制，本文在细胞水平进行实验。我们的实验结果表明，HBx能够通过抑制p53与IGFBP3启动子的结合，从而抑制IGFBP3的转录，进而抑制IGFBP3的促凋亡作用。本文揭示了HBx抑制细胞凋亡、导致HCC的新机制，丰富了对该领域的认识。
1 材料和方法 1.1 材料
1.1.1 细胞: 人肾上皮细胞293T由本实验室保存，培养在含有10%胎牛血清(购自PAA公司)的DMEM培养基中；人肝癌细胞HepG2、HBx转基因细胞HepG2-HBx由本实验室保存，HBV转基因细胞HepG2-C5、HepG2-4D14由解放军302医院徐东平教授惠赠。人结肠癌细胞HCT116-WT、HCT116-p53–/–来自中国医学科学院，培养在含有10%胎牛血清的DMEM培养基中。
1.1.2 质粒: IGFBP3-luc质粒为实验室所构建，IGFBP3-luc选取了IGFBP3启动子区–210?+60位(转录起始位点记为+1)的碱基片段[13]，载体为pGL2-Basic，酶切位点为NheⅠ和Hind Ⅲ。PCR引物详见表 1。
表 1. 实时定量PCR、ChIP Assay所用引物 Table 1. Primers for Real time-PCR and ChIP Assay
| Primers | Primer sequences (5'→3') |
| RT-IGFBP3-F | CTCTGCGTCAACGCTAGTGC |
| RT-IGFBP3-R | CGGTCTTCCTCCGACTCACT |
| RT-GAPDH-F | TGCACCACCAACTGCTTAG |
| RT-GAPDH-R | GATGCAGGGATGATGTTC |
| ChIP-F | GGTGGCCGGGCACACCTTGGT |
| ChIP-R | GGCTGCGGGGGCCCGTGCTTC |
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1.1.3 抗体及试剂: Myc抗体购自Sigma公司，tubulin、IGFBP3抗体购自CST公司，p53抗体购自Santa Cruz公司，荧光素酶检测试剂盒购自Promega公司，MLV逆转录试剂盒购自全式金公司，SYBR购自TOYOBO公司，ChIP Assay试剂盒购自碧云天公司，LipoPlus转染试剂购自Sage Creation公司。
1.2 质粒转染和蛋白质免疫印迹实验 将293T、HCT116-WT、HCT116-p53–/–细胞于转染前铺入相应的培养皿中，24 h后待细胞达到适宜的汇合度(293T 70%–80%、HCT116 30%–40%)时，进行转染。用无血清DMEM稀释转染试剂LipoPlus，使得转染试剂与DNA的比例为3 μL/μg，混匀后静置5 min，再与无血清DMEM稀释的质粒混合，静置20 min后加入细胞中。细胞置于5% CO2的培养箱中37 ℃培养，转染5 h后换液。培养48 h后收取细胞，弃培养基，加入1 mL PBS悬起细胞，1000 r/min、4 ℃离心5 min。弃上清，加入适量Lysis Buffer，冰上裂解15 min，12000 r/min、4 ℃离心10 min。上清转移至新EP管，加入2×Loading Buffer，煮样，进行SDS-PAGE，将蛋白转至PVDF膜上后进行Western Blot检测。
1.3 荧光素酶报告基因实验 293T、HCT116-WT细胞铺入24孔板，24 h后转染，pRL-TK质粒作为内参，空载体作为空白对照。24 h后收取细胞，根据Promega荧光素酶检测试剂盒的说明书进行检测。
1.4 RNA的提取及实时定量PCR实验 HepG2、HepG2-HBx、HCT116-WT、HCT116-p53–/–细胞转染48 h后，收取细胞，采用Trizol法提取总RNA，定量后采用MLV逆转录试剂盒中的Oligo dT将其反转录为cDNA。cDNA作为模板，根据需要采用相应引物(表 1)，进行实时定量PCR。
1.5 染色质免疫共沉淀(ChIP)实验 293T细胞共转Myc-p53及FLAG-HBx及空白对照48 h后，收取细胞，根据ChIP Assay试剂盒的说明书进行操作，使用Myc抗体进行免疫沉淀，分离与p53蛋白结合的DNA。已知p53与IGFBP3启动子的结合区域为–210?–159[13-14](转录起始位点记为+1)，我们选用位于–250?–110的片段作为引物，序列见表 1。最终产物用于实时定量PCR，检测HBx对p53与IGFBP3启动子区结合能力的影响。
2 结果和分析 2.1 HBV能够下调IGFBP3的mRNA及蛋白水平 首先，我们采用RNA-Deep-Sequencing技术分析HepG2和HBV转基因细胞HepG2-4D14中表达差异的基因。我们的研究重点主要在于HBV在原发性肝癌发生发展过程中所发挥的作用，因此在筛选HepG2和HBV转基因细胞HepG2-4D14中表达差异的基因时，重点分析了在细胞增殖、细胞凋亡以及肿瘤形成中发挥作用的基因。我们采用实时定量PCR技术在细胞水平进行验证，初步筛选基因。图 1-A展示了部分数据。IGFBP3的实时定量PCR验证结果与RNA-Deep-Sequencing结果一致，并且已有相关报道证实IGFBP3具有促凋亡作用，符合我们的研究目的，因此，我们优先选择了IGFBP3进行研究。
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| 图 1 HBV下调IGFBP3的mRNA及蛋白水平 Figure 1 HBx down-regulates the mRNA and protein level of IGFBP3. A: Real-time PCR was used to detect the mRNA level of KLF4, KLF7, IFI16, IGFBP3, TNFSF15 in HepG2 and HepG2-4D14 cells. The RNA level was normalized to GAPDH mRNA in cells. B: HepG2, HepG2-C5 and HepG2-4D14 cells were plated in 6-well plates and harvested after 48 h. The total RNAs were isolated with Trizol reagent, then mRNA of IGFBP3 was quantified by real-time PCR. The RNA level was normalized to GAPDH mRNA in cells. C: HepG2, HepG2-C5 and HepG2-4D14 cells were plated in 6-well plates and harvested after 48 h. The cell lysates were harvested for immunoblotting with IGFBP3 antibody. D: The total RNA of liver tissues of HBV transgenic and control mice was isolated with Trizol reagent, then mRNA of IGFBP3 was quantified by real-time PCR. |
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分析结果可知，与HepG2细胞相比，HepG2-4D14细胞中IGFBP3的mRNA水平下调了5倍。为了进一步证实该结果，我们采用实时定量PCR检测HBV转基因细胞HepG2-C5及HepG2-4D14细胞中IGFBP3的mRNA水平，如图 1-B所示，HBV转基因细胞HepG2-C5及HepG2-4D14细胞中IGFBP3的mRNA水平仅为HepG2细胞中的20%。我们采用IGFBP3抗体进行Western Blot，如图 1-C所示，在HBV转基因细胞HepG2-C5及HepG2-4D14细胞中IGFBP3的蛋白水平分别为HepG2细胞中的56%及46%。为了进一步确认，我们检测了HBV转基因小鼠肝组织中IGFBP3的mRNA水平，如图 1-D所示，HBV转基因小鼠肝组织中IGFBP3的mRNA水平仅为对照组小鼠的1/3。综上所述，HBV能够下调IGFBP3的mRNA及蛋白水平。
2.2 HBx抑制IGFBP3的转录 图 1证实HBV能够抑制IGFBP3的mRNA及蛋白水平，IGFBP3又是促凋亡因子，我们推测HBV通过抑制IGFBP3的表达从而抑制凋亡。IGFBP3的mRNA水平受到了抑制，因此我们认为相对于翻译后修饰，HBV更有可能是在转录阶段发挥作用。
为了确定HBV所编码的蛋白中能够抑制IGFBP3转录的蛋白，我们构建了IGFBP3启动子的报告基因系统，用以探索HBx抑制IGFBP3转录的机制。如图 2-A所示，HBx能够显著抑制IGFBP3的启动子活性，而HBs和HBc对IGFBP3的启动子活性没有明显的作用。因此，我们得知HBx是抑制IGFBP3转录的蛋白。为了进一步确定，我们选用HepG2和HBx转基因细胞HepG2-HBx (图 2-B)，采取实时定量PCR技术检测IGFBP3的mRNA水平。如图 2-B所示，HepG2-HBx细胞中IGFBP3的mRNA水平仅为HepG2细胞的25%，结果表明，HBx能够下调IGFBP3的表达。我们在293T及野生型HCT116细胞中转染Myc-HBx及IGFBP3-luc (图 2-C、图 2-D)，转染24 h后检测荧光素酶活性，如图 2-C、2-D所示，与对照组相比，过表达HBx的细胞中IGFBP3的荧光素酶活性显著减弱，仅为对照组的30%左右，这说明HBx能够抑制IGFBP3的启动子活性，从而抑制IGFBP3的转录。
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| 图 2 HBx抑制IGFBP3的转录 Figure 2 HBx inhibits the transcription of IGFBP3. A: 293T cells were plated in 24-well plates and transfected with FLAG-HBx, FLAG-HBs, FLAG-HBc, IGFBP3-luc and pRL-TK plasmids. Cells were harvested for luciferase assay after 24 h. The protein level of HBx, HBs, HBc and tubulin was detected by immunoblotting. B: HepG2, HepG2-HBx cells were plated in 6-well plates and harvested after 48 h. The total RNAs were isolated with Trizol reagent, then mRNA of IGFBP3 was quantified by real-time PCR. The RNA level was normalized to GAPDH mRNA in cells. The protein level of recombined HBx and tubulin were detected by immunoblotting. C: 293T cells were transfected with Myc-HBx, IGFBP3-luc and pRL-TK plasmids. Cells were harvested for luciferase assay after 24 h. Luciferase activity level was normalized to renilla activity. The protein level of HBx and tubulin was detected by immunoblotting. D: HCT116-WT cells were transfected with Myc-HBx, IGFBP3-luc and pRL-TK plasmids. Cells were harvested for luciferase assay after 24 h. Luciferase activity level was normalized to renilla activity. The protein level of HBx and tubulin was detected by immunoblotting. |
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2.3 p53促进IGFBP3的转录 为了确定p53在IGFBP3转录过程中发挥的作用，我们采用实时定量PCR检测IGFBP3在野生型HCT116细胞以及p53缺失的HCT116-p53–/–细胞(图 3-A)中的mRNA水平。如图 3-A所示，在HCT116-p53–/–细胞中，IGFBP3的mRNA水平显著低于野生型HCT116细胞，仅为野生型细胞的24%。我们在293T及野生型HCT116细胞中转染Myc-p53 (图 3-B、3-C)，以确定p53对IGFBP3启动子活性的影响。如图 3-B、3-C所示，过表达的p53能够显著促进IGFBP3的启动子活性，与对照相比有2倍左右的上调。综上所述，p53通过促进IGFBP3的启动子活性来促进IGFBP3的转录。
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| 图 3 p53促进IGFBP3的转录 Figure 3 p53 promotes the transcription of IGFBP3. A: HCT116-WT and HCT116-p53–/– cells were plated in 6-well plates and harvested for real-time RCR after 48 h. The protein level of p53 was detected by immunoblotting. B: 293T cells were plated in 24-well plates and transfected with Myc-p53, IGFBP3-luc and pRL-TK plasmids. Cells were harvested for luciferase assay after 24 h. The protein level of p53 and tubulin was detected by immunoblotting. C: HCT116-WT cells were plated in 24-well plates and transfected with Myc-p53, IGFBP3-luc and pRL-TK plasmids. Cells were harvested for luciferase assay after 24 h. The protein level of p53 and tubulin was detected by immunoblotting. |
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2.4 HBx对IGFBP3转录的抑制依赖于p53 根据上述实验结果，我们提出假设，p53可能会介导HBx对IGFBP3的抑制作用。为了探究HBx对IGFBP3转录的抑制是否依赖于p53，我们在野生型HCT116细胞及HCT116-p53–/–细胞中转染Myc-HBx，转染48 h后采用实时定量PCR检测IGFBP3 mRNA水平的变化，Western Blot检测Myc-HBx蛋白的表达情况(图 4-A)。如图 4-B所示，在野生型HCT116细胞中转染HBx后，IGFBP3的mRNA水平与对照相比，有80%的下调，而在HCT116-p53–/–的细胞中，IGFBP3的mRNA水平与对照相比没有明显变化，因此，HBx对IGFBP3的抑制作用依赖于p53。
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| 图 4 HBx对IGFBP3转录的抑制依赖于p53 Figure 4 The inhibition effect of HBx on IGFBP3 transcription depends on p53. HCT116-WT and HCT116-p53–/– cells were transfected with Myc-HBx plasmids. A: cells were harvested after 48 h. The protein level of p53 and tubulin was detected by immunoblotting. B: the total RNAs were isolated with Trizol reagent, then mRNA of IGFBP3 was quantified by real-time PCR. The RNA level was normalized to GAPDH mRNA in cells. |
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2.5 HBx通过影响p53与IGFBP3启动子的结合抑制IGFBP3的转录 已有文献报道，HBx能够抑制p53与靶基因启动子的结合[7]。我们推测HBx可能抑制p53与IGFBP3启动子的结合。我们在293T细胞中转染FLAG-HBx和Myc-p53，采用染色质免疫共沉淀技术以及实时定量PCR，检测p53结合IGFBP3启动子的能力。如图 5-B所示，我们使用Myc抗体进行免疫沉淀，在沉淀获得等量p53蛋白(图 5-A)的情况下，转染HBx的细胞中p53与IGFBP3启动子的结合显著减弱，与对照相比，结合的启动子含量降至30%，说明HBx能够显著抑制p53与IGFBP3启动子的结合。这可能是HBx抑制IGFBP3转录的机制之一。
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| 图 5 HBx通过影响p53与IGFBP3启动子的结合抑制IGFBP3的转录 Figure 5 HBx inhibits the binding of p53 to the promoter of IGFBP3. 293T cells were trasfected with Myc-p53 and FLAG-HBx. Cells were harvested after 48 h and subjected to ChIP Assay. A: the protein level of p53, HBx and tubulin was detected by immunoblotting. B: Real-time PCR was performed to quantify the amount of p53 binding DNA. |
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3 讨论 HBV的慢性感染是诱发肝癌的主要原因。HBx对HBV的复制至关重要，同时也在HCC发展过程中发挥作用。HBx是多功能蛋白，参与调控基因转录[3]、细胞增殖、DNA损伤修复，促进细胞周期[15]、激活细胞内信号转导通路[4]、调控细胞凋亡[5]。例如，HBx能够通过抑制线粒体膜上死亡配体Bid的表达，抑制线粒体释放细胞色素C，降低caspase-8和caspase-3的活性，进而抑制凋亡，促进肝癌的发生[16]。因此，对HBx作用方式的研究能够帮助阐明HCC的发生发展机制。
已有报道指出HBx能够通过招募组蛋白去乙酰化酶(HDAC1)，促进其对IGFBP3启动子区转录因子Sp1的去乙酰化，进而抑制IGFBP3的转录[17]。该文发现HBx能够直接与HDAC1结合并且这种结合作用不依赖于p53。本文发现HBx通过p53依赖性方式抑制IGFBP3的转录。关于2项研究所得结论的不同，我们进行了如下分析。该文使用Hep3B细胞、选用IGFBP3启动子区–604?+24 nt的片段，过表达p53进行免疫沉淀实验，发现梯度过表达p53不影响HBx与HDAC1结合，推知该结合过程不依赖于p53。我们的实验使用野生型及p53缺失的HCT116细胞、选用IGFBP3启动子区–210?+60 nt的片段[13]，转染48 h后进行实时定量PCR，检测HBx对IGFBP3转录的影响，结果表明在缺失p53的情况下，HBx不能抑制IGFBP3的转录。我们还通过染色质免疫沉淀技术发现HBx抑制p53与IGFBP3启动子的结合，从而抑制IGFBP3的转录。综合两项研究，我们认为，IGFBP3的启动子区存在多个蛋白的结合位点，HBx能够通过不同的机制抑制IGFBP3的转录，p53在各个机制中发挥的作用也不尽相同。同时，细胞及启动子片段的选择、细胞培养的时间、研究方法的不同也会导致结论的不同。另外，该文没有检测p53对IGFBP3总体转录情况的作用，与我们的结论并不矛盾。
本实验首次发现p53在HBx抑制IGFBP3转录过程中的作用，HBx通过干扰p53与IGFBP3启动子的结合进而抑制IGFBP3的转录，揭示了HBx促进肝癌发生的新机制。本实验对HBV调控IGFBP3机制的研究能够为肿瘤的治疗及药物的开发奠定基础。
References
| [1] | Lucifora J, Xia YC, Reisinger F, Zhang K, Stadler D, Cheng XM, Sprinzl MF, Koppensteiner H, Makowska Z, Volz T, Remouchamps C, Chou WM, Thasler WE, Hüser N, Durantel D, Liang TJ, Munk C, Heim MH, Browning JL, Dejardin E, Dandri M, Schindler M, Heikenwalder M, Protzer U. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science, 2014, 43(6176): 1221-1228. |
| [2] | Seeger C, Mason WS. Molecular biology of hepatitis B virus infection. Virology, 2015, 479-480: 672-686. DOI:10.1016/j.virol.2015.02.031 |
| [3] | Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Science, 2006, 97(10): 977-983. DOI:10.1111/cas.2006.97.issue-10 |
| [4] | Feng GX, Li J, Zheng MY, Yang Z, Liu YX, Zhang SQ, Ye LH, Zhang WY, Zhang XD. Hepatitis B virus X protein up-regulates C4b-binding protein α through activating transcription factor Sp1 in protection of hepatoma cells from complement attack. Oncotarget, 2016, 7(19): 28013-28026. DOI:10.18632/oncotarget.v7i19 |
| [5] | Kongkavitoon P, Tangkijvanich P, Hirankarn N, Palaga T. Hepatitis B virus HBx activates Notch signaling via Delta-like 4/Notch1 in hepatocellular carcinoma. PLoS One, 2016, 11(1): e0146696. DOI:10.1371/journal.pone.0146696 |
| [6] | Chao CCK. Inhibition of apoptosis by oncogenic hepatitis B virus X protein:implications for the treatment of hepatocellular carcinoma. World Journal of Heptatology, 2016, 8(25): 1061-1066. DOI:10.4254/wjh.v8.i25.1061 |
| [7] | Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity and association with transcription factor ERCC3. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(6): 2230-2234. DOI:10.1073/pnas.91.6.2230 |
| [8] | Chung TW, Lee YC, Ko JH, Kim CH. Hepatitis B Virus X protein modulates the expression of PTEN by inhibiting the function of p53, a transcriptional activator in liver cells. Cancer Research, 2003, 63(13): 3453-3458. |
| [9] | Lee SG, Rho HM. Transcriptional repression of the human p53 gene by hepatitis B viral X protein. Oncogene, 2000, 19(3): 468-471. DOI:10.1038/sj.onc.1203312 |
| [10] | Nguyen DV, Li Calzi S, Shaw LC, Kielczewski JL, Korah HE, Grant MB. An ocular view of the IGF-IGFBP system. Growth Hormone& IGF Research, 2013, 23(3): 45-52. |
| [11] | Hwa V, Oh Y, Rosenfeld RG. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocrine Review, 1999, 20(6): 761-787. |
| [12] | Mohan S, Baylink DJ. IGF-binding proteins are multifunctional and act via IGF-dependent and -independent mechanisms. Journal of Endocrinology, 2002, 175(1): 19-31. DOI:10.1677/joe.0.1750019 |
| [13] | Hanafusa T, Shinji T, Shiraha H, Nouso K, Iwasaki Y, Yumoto E, Ono T, Koide N. Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation. BMC Cancer, 2005, 5(1): 9. DOI:10.1186/1471-2407-5-9 |
| [14] | Bourdon1 JC, Degui-Chambon V, Lelong JC, Dessen P, May P, Debuire B, May E. Further characterisation of the p53 responsive element-identification of new candidate genes for trans-activation by p53. Oncogene, 1997, 14(1): 85-94. DOI:10.1038/sj.onc.1200804 |
| [15] | Kim HS, Lee WJ, Lee SW, Chae HW, Kim DH, Oh Y. Insulin-like growth factor binding protein-3 induces G1 cell cycle arrest with inhibition of cyclin-dependent kinase 2 and 4 in MCF-7 human breast cancer cells. Hormone and Metabolic Research, 2010, 42(3): 165-172. DOI:10.1055/s-0029-1243190 |
| [16] | Lee YI, Hwang JM, Im JH, Lee YI, Kim NS, Kim DG, Yu DY, Moon HB, Park SK. Human hepatitis B virus-X protein alters mitochondrial function and physiology in human liver cells. The Journal of Biological Chemistry, 2004, 279(15): 15460-15471. DOI:10.1074/jbc.M309280200 |
| [17] | Shon JK, Shon BH, Park IY, Lee SU, Fa L, Chang KY, Shin JH, Lee YI. Hepatitis B virus-X protein recruits histone deacetylase 1 to repress insulin-like growth factor binding protein 3 transcription. Virus Research, 2009, 139(1): 14-21. DOI:10.1016/j.virusres.2008.09.006 |
