焦童1,2, 刘宁宁1, 叶昕1
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![yex@im.ac.cn](http://journals.im.ac.cn/html/actamicrocn/2017/12/images/REemail.gif)
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
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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。
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