乙肝病毒调控hTERT及ZHX2在肝细胞肝癌发生过程中的作用及机制研究
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摘要
目的
肝细胞肝癌(hepatocellular carcinoma,HCC)是严重危害人类生命的恶性肿瘤,全世界每年新发病例100万,中国是HCC的高发地区,病例数约占全球HCC病例的55%。乙型肝炎病毒(hepatitis B virus,HBV)感染是HCC发生的主要危险因素,临床研究发现63.2%的肝癌患者乙肝表面抗原阳性。因此,揭示HBV相关HCC分子发病机制极为重要和迫切。
HBV相关HCC的发生是多基因、多步骤、多阶段的过程,HBV DNA可以直接整合到宿主细胞基因组发挥其顺式激活作用,也可以通过其编码蛋白发挥反式激活作用。乙肝病毒及其蛋白对宿主多种基因,尤其是肿瘤相关基因的调控,在HCC发生中发挥重要作用。寻找受HBV调控的宿主关键基因并阐明其机制,不仅有助于揭示HBV相关HCC发生机制,还将为HCC治疗提供新靶点。
本研究在前期基础上,结合当今研究进展,选择两种肝癌相关重要宿主基因:端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)及新型转录抑制因子ZHX2(zinc-finger and homeoboxe 2),深入研究HBV对它们的调控作用及其在HCC发生中的作用。
端粒酶的活化和端粒长度的维持与肝细胞永生化密切相关。HBV相关HCC中,100%端粒酶呈激活状态,且端粒酶逆转录酶hTERT表达上调。本室前期研究发现,肝癌细胞系HepG2中过表达HBV反式激活蛋白preS2可以上调hTERT表达,提示preS2介导的hTERT激活是HBV相关HCC发生的重要机制,本研究将在此基础上进一步阐明其机制。
甲胎蛋白(alpha fetoprotein,AFP)是HCC临床检测金指标,在临床上已广泛应用。但该检测还不能完全满足临床要求,所以寻找有助于早期诊断的肝癌特异性标记物具有重要理论意义和临床应用价值。磷脂酰肌醇蛋白聚糖(Glypican3,GPC3)是近年新兴的具有潜力的肝癌标记物。
ZHX2是2003年发现的新型转录抑制因子,广泛存在于脊椎动物体内。目前已知小鼠中受到ZHX2负调控的基因仅有3种,即AFP、H19和GPC3,此三种基因均具有肝癌特异性高表达的特点:在胚胎期高表达,出生后受到抑制,而在肝癌发生时再次被激活。且AFP和GPC3具有癌基因特性,参与HCC的发生。本室前期研究证明ZHX2抑制肝癌细胞系中AFP的表达,提示ZHX2在肝癌发生中具有重要作用。已有报道证明,HCC患者中AFP表达与HBV感染密切相关,但在HCC中HBV是否可以调控ZHX2,以及ZHX2是否调控人肝癌中GPC3的表达,迄今尚未见报道,本研究将对此进行系列研究。
综上,为了深入探讨HBV相关HCC的发病机制,本研究在前期基础上,选择hTERT及ZHX2作为HCC发生的关键基因,拟从以下几个方面进行研究:
1.HBV编码的preS2蛋白反式激活hTERT参与HCC发生的分子机制。
2.HBV对肝癌相关新型转录抑制因子ZHX2的调控及作用研究:
(1)HBV对HCC中ZHX2的调控作用。
(2)ZHX2对HCC中GPC3的抑制作用。
方法
第一部分HBV编码的preS2蛋白反式激活hTERT参与HCC发生的分子机制
1.preS2对hTERT的调控作用
1.1细胞实验:设计合成针对preS2的反义寡核苷酸,脂质体转染HepG2.2.15细胞,RT-PCR、Western blot检测反义封闭preS2后hTERT表达。
1.2临床检测:收集HCC组织标本16例,RT-PCR、Western blot分别检测preS2和hTERT表达。
2.preS2蛋白对hTERT启动子反式激活作用的研究
2.1 preS2-EGFP融合蛋白在肝癌细胞中的表达:构建preS2-EGFP融合蛋白表达载体,脂质体转染HepG2细胞,48h后进行DAPI核染,荧光显微镜观察preS2的表达定位情况。
2.2 preS2对hTERT启动子的激活作用:preS2表达载体(连有HA标签)与hTERT启动子报告质粒共转染肝永生化细胞LO2、肝癌细胞HepG2、BEL7402以及非洲绿猴肾细胞COS-7,双荧光素酶报告基因检测preS2对hTERT启动子的作用;同时在HepG2细胞中以不同剂量preS2表达载体与hTERT启动子报告质粒共转染,验证preS2对hTERT作用的剂量依赖性。
3.preS2反式激活hTERT启动子的分子机制研究
3.1 preS2介导的hTERT启动子激活中c-myc的表达:肝癌细胞HepG2转染preS2表达载体(连有HA标签)后,Western blot检测c-myc蛋白的表达变化。
3.2 preS2激活hTERT启动子关键区段(PRR)的确定:构建系列截短、缺失及突变的hTERT启动子报告质粒,与preS2表达载体共转染HepG2和COS-7,双荧光素酶报告基因检测,确定preS2在hTERT启动子上的作用靶点,命名为preS2-Resiponsible Region,简称PRR。
3.3 EMSA实验:
3.3.1细胞模型:肝癌细胞HepG2转染preS2表达载体后,两步法提取核蛋白。
3.3.2临床HCC组织核蛋白抽提:收集10例preS2阳性和6例阴性新鲜肝癌组织标本,常规抽提核蛋白。
3.3.3 EMSA实验:合成PRR寡核苷酸片段,地高辛标记后作为探针,分别与上述细胞核蛋白和组织核蛋白进行EMSA,检测preS2与PRR的结合。
第二部分HBV对HCC中ZHX2的调控及其作用研究
1.HBV对HCC中ZHX2的调控作用
1.1细胞研究
1.1.1 HBV及其基因片段过表达对HepG2细胞中ZHX2表达的作用:HBV及其编码蛋白HBX、HBC、preS2表达质粒(空载体为对照)脂质体转染HepG2细胞,48h后收集细胞,RT-PCR、Western blot检测ZHX2表达变化。
1.1.2反义抑制HBV不同片段对HepG2.22.15细胞中ZHX2表达的作用:设计合成针对HBX、HBC、preS2的反义寡核苷酸,脂质体转染HepG2.2.15细胞(无关序列转染组为对照),48h后收集细胞,RT-PCR、Western blot检测ZHX2表达变化。
1.2动物实验
6月龄HBV转基因小鼠2只、正常BALB/c对照小鼠3只,取肝组织提取总RNA,实时定量RT-PCR检测两组小鼠肝组织中ZHX2表达差异。
1.3临床检测
收集33例HCC组织标本,按HBV感染情况分为两组,RT-PCR检测两组标本中ZHX2表达差异。
2.ZHX2对HCC中GPC3的抑制作用及其机制的初步探讨
2.1 ZHX2对不同肝癌细胞系中GPC3的抑制作用研究
2.1.1不同肝癌细胞系中ZHX2与GPC3表达的相关性:培养肝癌细胞系HepG2、Hep3B、SMMC7721及肝永生化细胞系LO2,收集对数生长期细胞,RT-PCR检测ZHX2及GPC3的mRNA表达情况。
2.1.2过表达实验:在ZHX2相对低表达的细胞系(HepG2、Hep3B)中,脂质体转染ZHX2表达载体,48h后收集细胞,RT-PCR、Western blot检测GPC3的表达变化。
2.1.3 siRNA基因沉默实验:在ZHX2相对高表达的细胞系(SMMC7721、LO2)中,脂质体转染针对ZHX2的siRNA质粒,干扰ZHX2的表达,48h后收集细胞,RT-PCR检测GPC3的表达变化。
2.1.4去甲基化恢复ZHX2表达对GPC3的影响:不同剂量去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理HepG2细胞恢复ZHX2的表达,RT-PCR、Westernblot检测GPC3的表达变化。
2.2 HCC临床标本中ZHX2与GPC3表达的相关性研究
收集33例HCC组织标本,Western blot检测标本中ZHX2、GPC3表达情况,采用Spearman相关系数分析ZHX2与GPC3表达的关系。
2.3 ZHX2抑制GPC3启动子活性研究
2.3.1 GPC3核心启动子报告质粒载体的构建:设计引物,PCR扩增GPC3转录起始点上游218bp片段,克隆入pGL3-Basic载体,构建GPC3核心启动子荧光素酶报告基因载体pGL3-GPC3-218,分别在HepG2和COS-7细胞中通过基因转染和双荧光素酶报告基因检测验证启动子活性。
2.3.2 ZHX2对GPC3启动子的抑制作用研究:不同剂量ZHX2表达载体与GPC3核心启动子报告质粒共转染HepG2细胞,双荧光素酶报告基因检测ZHX2对GPC3核心启动子的作用。
结果
第一部分HBV编码的preS2蛋白对hTERT反式激活作用的分子机制
1.preS2蛋白对HCC中hTERT表达的调控作用
1.1反义封闭preS2抑制HepG2.2.15细胞中hTERT表达
本室前期研究发现,preS2表达质粒瞬时转染细胞可以明显上调HepG2细胞中hTERT的表达,提示preS2可以增强hTERT表达。为克服基因过表达的人为因素影响,本研究设计合成针对preS2的反义寡核苷酸片段,脂质体转染HepG2.2.15细胞(整合有HBV基因组,可表达HBV蛋白),48h后,收集细胞,RT-PCR、ELISA结果显示反义寡核苷酸可有效降低preS2 mRNA和细胞上清中HBsAg的表达;同时,HepG2.2.15细胞中反义封闭preS2的表达后,hTERT在mRNA和蛋白水平的表达均明显降低。上述结果显示:封闭preS2蛋白可以抑制肝癌细胞系中hTERT表达。
1.2 preS2阳性HCC组织标本中hTERT表达显著高于preS2阴性HCC标本
为进一步研究HCC中preS2对hTERT的调控作用,本研究收集16例HCC组织标本,检测其preS2及hTERT的表达。RT-PCR结果显示:16例HCC标本中,10例HBV阳性的HCC标本其preS2亦呈阳性表达,6例HBV阴性的HCC标本其preS2亦呈阴性表达;Western blot检测显示:10例preS2阳性表达的HCC标本hTERT的表达水平明显高于6例preS2阴性表达的HCC标本(p＜0.05)。上述结果提示,临床HCC标本中preS2表达与hTERT表达正相关,与细胞系中的研究结果相符。
2.preS2蛋白在不同细胞中反式激活hTERT启动子
众多文献证实,hTERT的转录水平调控是端粒酶表达调控的主要方式,为此,本研究构建preS2真核表达载体,研究其对hTERT启动子的调控作用。
2.1 preS2融合蛋白定位表达于肝癌细胞核内
为进一步研究preS2对hTERT的调控作用,本研究成功构建preS2绿色荧光融合蛋白表达载体,命名为pEGFP-preS2;转染HepG2细胞48h后DAPI核染,荧光显微镜观察绿色荧光主要定位于细胞核,显示preS2可在肝癌细胞中表达,并主要表达于细胞核,具有转录因子的特点。
2.2 preS2在不同细胞中反式激活hTERT启动子
pEGFP-preS2与hTERT启动子报告质粒共转染不同细胞系,双荧光素酶报告基因检测结果显示:在肝永生化细胞LO2、肝癌细胞HepG2、BEL7402以及非洲绿猴肾细胞COS-7中,preS2对hTERT启动子均具有明显的反式激活作用,与pcDNA3空载体转染组相比,作用强度分别为2.469倍、2.933倍、2.556倍、2.426倍,提示preS2对hTERT启动子的反式激活作用并非肝细胞特异性;不同剂量preS2表达载体与全长hTERT启动子报告质粒共转染HepG2,双荧光素酶报告基因检测显示:preS2对hTERT启动子反式激活作用具有剂量依赖性。
3.preS2反式激活hTERT启动子分子机制研究
3.1 preS2反式激活hTERT启动子不依赖于c-myc
hTERT启动子区存在2个c-myc结合位点,且文献证实多种病毒通过该位点激活hTERT。然而,本研究中Western blot检测显示:过表达preS2并没有影响c-myc蛋白的表达变化,提示preS2对hTERT启动子的反式激活是不依赖c-myc途径的。
3.2 preS2反式激活hTERT关键区段PRR的确定
为确定preS2激活hTERT的关键片段,本研究成功构建系列截短、缺失及突变的hTERT启动子报告质粒,分别命名为pGL3B-895、pGL3B-371、pGL3B-306、pGL3B-349、pGL3B-329、pGL3B-318、pGL3B-DELS2、pGL3B-MUT371R、pGL3B-MUT349S。上述载体分别与preS2表达载体共转染HepG2和COS-7细胞,双荧光素酶报告基因检测结果分析,将preS2在hTERT启动子上的作用范围确定在-371～-307之间65bp区域,并进一步缩短至-349～-329bp,命名为preS2 responsive-region,简称PRR。
3.3 EMSA分析preS2与PRR的结合
合成PRR寡核苷酸,地高辛标记后与preS2转染的HepG2细胞核蛋白共孵育,凝胶电泳迁移抑制实验(EMSA)结果显示:preS2表达载体转染的核蛋白可与PRR探针结合,此种结合可被特异性PRR片段竞争性抑制,且HA抗体作用后电泳带明显后滞,提示preS2可与hTERT启动子上PRR位点特异性结合。为进一步验证上述结果,收集并抽提HCC组织标本核蛋白,EMSA结果显示:preS2阳性的HCC标本与PRR的结合能力明显强于preS2阴性的HCC标本,与细胞实验结果一致。
第二部分HBV对HCC中ZHX2的调控及其作用研究
ZHX2是2003年发现的新型转录抑制因子,迄今在小鼠中仅发现了三个受ZHX2调控的靶基因:AFP、GPC3及H19。其中,H19为印记基因,而AFP和GPC3均具有肝癌特异性高表达的特点,并参与肝癌发生。本室前期研究发现,ZHX2抑制肝癌细胞系中AFP表达,提示其在肝癌中的重要作用。本部分实验首次探讨HBV对ZHX2的调控作用,并探讨了ZHX2对人肝癌细胞中GPC3的抑制作用,本部分研究将为阐明HBV相关HCC的发生机制提供新的实验依据。
1.HBV对HCC中ZHX2表达的调控作用
本研究中利用细胞模型、转基因动物及临床检测,系统研究HBV对ZHX2的调控作用。
1.1 HBV抑制肝癌细胞系中ZHX2的表达
为研究HBV对ZHX2的抑制作用,分别进行ZHX2过表达实验和反义寡核苷酸抑制实验。RT-PCR、Western blot检测结果显示:肝癌细胞SMMC7721中过表达HBV及其编码蛋白HBX、HBC、preS2后,ZHX2表达下调,其中以HBV和HBC的影响更为明显;HepG2.2.15细胞中反义封闭HBX、HBC、preS2表达后,ZHX2表达上调,其中封闭HBC表达后ZHX2表达上调更为明显。上述结果提示,HBV及其基因片段可以抑制肝癌细胞中ZHX2表达,且HBC抑制作用最强。
1.2 HBV转基因鼠肝组织中ZHX2表达显著低于对照鼠
HBV转基因鼠肝组织稳定表达HBV及其抗原,是研究HBV感染的良好动物模型。本研究选择6月龄HBV转基因小鼠,并与正常同系BALB/c小鼠为对照,抽提肝组织RNA,实时定量RT-PCR检测ZHX2表达。结果显示:HBV转基因小鼠肝组织中ZHX2表达明显低于正常对照组。(p＜0.001)
1.3 HBV阳性HCC标本中ZHX2 mRNA显著低于HBV阴性组
为进一步验证HBV对ZHX2的抑制作用,收集33例HCC组织标本,RT-PCR检测ZHX2表达,结果显示:HBV阳性组标本中ZHX2 mRNA的表达水平明显低于HBV阴性组。(p＜0.01),与预期一致。
2.ZHX2对HCC中GPC3的抑制作用及其机制的初步探讨
2.1 ZHX2抑制人肝癌细胞系中GPC3的表达
RT-PCR结果显示,四种肝癌细胞系中ZHX2与GPC3表达负相关:在ZHX2相对低表达的细胞系HepG2、Hep3B中GPC3相对高表达;在ZHX2相对高表达的细胞系SMMC7721、LO2中GPC3相对低表达。
ZHX2转染实验结果表明,HepG2、Hep3B细胞系中过表达ZHX2后,GPC3的表达水平降低;siRNA基因沉默实验证明,在SMMC7721、LO2细胞系中干扰ZHX2的表达后,GPC3的表达水平显著升高。
有研究显示,甲基化可以抑制ZHX2表达,为进一步研究ZHX2对GPC3的调控作用,利用不同浓度去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理HepG2细胞(ZHX2低表达)。RT-PCR、Western blot检测结果显示:去甲基化可以升高ZHX2的表达水平;且随着去甲基化药物浓度的升高,GPC3的表达降低。
上述结果均提示,ZHX2可以抑制人肝癌细胞系中GPC3的表达。
2.2 HCC组织标本中ZHX2表达与GPC3负相关
为进一步验证HCC标本中ZHX2对GPC3的调控作用,收集33例HCC组织标本,Western blot检测ZHX2和GPC3表达后,半定量分析其表达水平,并进行Spearman相关系数分析,结果显示:ZHX2与GPC3呈中度负性相关关系且具有统计学意义(r=-0.4968,p＜0.01)。
2.3 ZHX2明显抑制GPC3核心启动子活性
2.3.1 GPC3核心启动子荧光素酶报告基因载体的构建
PCR扩增GPC3基因转录起始点上游218bp片段,克隆入pGL3-Basic质粒,构建GPC3核心启动子荧光素酶报告基因载体。基因转染实验及双荧光素酶报告基因检测结果显示:所构建的GPC3核心启动子报告质粒在HepG2和COS-7细胞中均具有启动子活性。
2.3.2 ZHX2抑制GPC3核心启动子活性
ZHX2表达载体与GPC3核心启动子报告质粒共转染HepG2细胞,双荧光素酶报告基因检测结果显示:ZHX2对GPC3核心启动子具有明显抑制作用(p＜0.01)。
结论
1.细胞系反义封闭实验和临床标本检测进一步验证HBV编码的preS2蛋白对hTERT的激活作用。共转染双荧光素酶报告基因检测和EMSA实验证实preS2蛋白通过作用于hTERT启动子的PRR区域(位于hTERT转录起始点上游-349～-329bp),反式激活hTERT进而活化端粒酶,从而参与HCC的形成。
2.从细胞系过表达和反义封闭实验、HBV转基因小鼠动物模型以及临床标本检测分析三方面证实:在HCC中,HBV可以抑制新型转录抑制因子ZHX2的表达,进而参与HCC发生和发展。
3.细胞系过表达和小干扰实验以及临床标本检测分析证实:ZHX2抑制HCC中GPC3的表达;成功构建人GPC3基因核心启动子报告质粒,共转染及双荧光素酶报告基因检测显示ZHX2可有效抑制GPC3启动子活性,在转录水平进一步验证了ZHX2对GPC3的表达抑制作用。
创新性和意义
1.本研究提出HBV编码的preS2蛋白通过调控端粒酶活性,进而参与细胞恶性变的论点,为阐明HBV相关肝细胞肝癌致病机制提供新思路。发现preS2反式激活hTERT的关键靶点,为HBV活化端粒酶提供直接证据。
2.众多研究提示ZHX2是HCC中一种潜在的抑癌基因。本研究首次证实HBV在HCC发生中抑制ZHX2的表达,为阐明HBV相关HCC的发病机制提供新的思路。
3.本研究首次证实在HCC中ZHX2对GPC3的抑制作用,并报道人ZHX2对GPC3启动子的抑制作用,为进一步探讨ZHX2对GPC3抑制作用的分子机制以及发现新的肝癌标志物奠定基础。
OBJECTIVES
Hepatocellular carcinoma(HCC) is a kind of malignant tumor that affects human health severely.China is HCC high-incidence district and takes up 55%of HCC cases in the world.HCC incidence in developed countries is increasing in recent years. Chronic hepatitis B virus(HBV) infection has been identified as a leading risk factor for HCC in China.So it is urgent to reveal the mechanisms of HBV related HCC.
The mechanism of HBV related HCC is complicated.The integration of HBV-DNA into the host genome can trigger cis-activation directly;Also regulatory proteins HBX and preS2 activators encoded by integrated subviral HBV genomes can exert a tumor promoter-like function,resulting in positive selection of cells producing a functional regulatory protein.To search the key genes regulated by HBV,not only provides newsights into mechanisms of HBV related HCC development,but also gives new potential targets for drug design.
Here,two important HCC related genes,human telomerase reverse transcriptase (hTERT) and zinc-finger and homeoboxe 2(ZHX2) were selected to be further studied.
It has been proved that telomerase activation is an important event during the process of immortalization and upregulation of hTERT might be a critical event in the development of human cancers,including HCC.Earlier studies from our laboratory have shown that preS2 overexpression can upregulate both hTERT expression and telomerase activity in HepG2 cells,resulting in enhanced cell growth which could be abolished by knocking down of hTERT in the cell,indicating that preS2 involves in HCC via hTERT upregulation.However,what's the mechanism remains unknown.In the first part of the study,cotransfection and report assay were involved to explore the molecular mechanism of preS2-mediated hTERT upregulation.
Zinc-finger and homeoboxes 2(ZHX2) gene belongs to a small family of transcription factors that also includes ZHX1 and ZHX3.All ZHX proteins contain two C2-H2 zinc-finger motifs and four homeodomains,are ubiquitously expressed and localized to the nuclei of cells,and appear to function as transcriptional repressors. Insight into the biological function of ZHX2 has come from the analysis of alpha-fetoprotein(AFP) expression in BALB/cJ mice.ZHX2 is reported to be responsible for adult AFP repression in BALB/cJ mice.H19 and Glypican 3 are two additional targets of ZHX2 in the mice liver and have the same expression law as AFP. Our previous studies show that ZHX2 is a repressor of AFP in HCC cell lines,which indicates the potential role of ZHX2 in HCC development.In the second part of this study,we aim to explore if HBV regulate ZHX2 and if ZHX2 is responsible for GPC3 regulation in HCC.
In order to investigate the mechanisms of HBV related HCC and gene regulation during HCC development,we focus our studies on the following aspects:
1.The precise mechanism for preS2 mediated transcriptional activation of the hTERT gene in HCC development.
2.The role of ZHX2 in HBV related HCC:
(1) The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC.
(2) The regulation of ZHX2 on GPC3 expression in HCC and the mechanism.
METHODS
1.The molecular mechanism for preS2 mediated transcriptional activation of the hTERT in HCC development
(1) HepG2.2.15 cells were pre-treated with preS2 antisense RNA to block the expression of preS2,RT-PCR and Western blot were applied to detect the expression change of hTERT.
(2) 16 HCC samples were collected,RT-PCR and Western blot were applied to detect the expression of preS2 and hTERT respectively.
(3) preS2-EGFP fusion protein eukaryotic expression vector was constructed and named pEGFP-preS2.48h After transfected into HepG2 cells,the cells were stained with 4',6-diamidino-2-phenylindole(DAPI).Digital images were taken from a OlympusⅨ81 fluorescence microscope with 400×magnification.
(4) 0.5μg hTERT luciferase reporter and 0.5μg preS2 expression plasmids were cotransfected into HepG2/BEL7402/LO2/COS-7.Meanwhile,different dose preS2 expression plasmids were used in HepG2.20ng SV40-Renilla was used as control to standardize the transcription efficiency.Cells were harvested 48h later in reporter lysis buffer.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocol.
(5) HepG2 cells were transfected with pcS2-HA expression plasmids(preS2 gene fused with hemaglutinin tag) or pcDNA3.Western blot was performed to detect the expression change of c-myc.
(6) To define the region of the hTERT promoter that was necessary for the preS2 response,series truncated/deleted/mutated hTERT promoter reporter plasmids were constructed.Luciferase assays were performed after cotransfected with pcS2-HA in HepG2 and COS-7.
(7) In order to further explore if preS2 could bind with PRR,EMSA were involved with nuclear extract from both HepG2 cells transfected with pcS2-HA and freshly isolated HCC samples.
2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC
(1) HBV and its viral proteins HBX/HBC/preS2 were overexpressed in HepG2 cells. Antisense RNA against HBX/ HBC/ preS2 were involved in HepG2.2.15. RT-PCR and Western blot were applied to detect the expression change of ZHX2.
(2) 33 HCC samples were grouped by HBV infection status,RT-PCR was applied to detect the expression difference of ZHX2;HBV DNA loads were detected by real-time quantitative PCR with the HBV-positive HCC tissue genomic DNA, ZHX2 expression levels were analyzed with HBV DNA.
(3) Total RNA of the liver tissues were extracted by TRIZOL from 2 HBV transgene mice and 3 normal BALB/c.All the mice were 6 months old.ZHX2 expression levels were determined by real-time quantitative PCR.
3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism
(1) RT-PCR was applied to detect the different expression levels of ZHX2 and GPC3 in HCC cell lines HepG2、Hep3B、SMMC7721 and immortalized liver cell line LO2.ZHX2 was overexpressed in cell lines express low ZHX2 levels.In contrast, siRNA-mediated ZHX2 knockdown was applied in cell lines express high ZHX2 levels.Then RT-PCR and Western blot were applied to detect the expression change of GPC3.
(2) HepG2 cells were treated with 5-Aza-CdR of different dose to recover ZHX2 expression.RT-PCR and Western blot were applied to detect the expression change of GPC3.
(3) 33 HCC samples were collected.ZHX2 and GPC3 expression were detected by Western blot.The relationship of ZHX2 and GPC3 expression was analyzed by Spearman analysis.
(4) GPC3 core promoter reporter plasmid was constructed and named pGL3-GPC3-218.The promoter activity was validated in HepG2 and COS-7 by Dual-Luciferase Reporter Assay.Different dose of pcDNA3-ZHX2-HA(ZHX2 expression vector with HA tag) were cotransfected with pGL3-GPC3-218. pRL-TK plasmids were involved in each group to normalize transfection efficiency.Cells were harvested 48h later for luciferase assay.
RESULTS
1.The molecular mechanism for preS2-mediated transcriptional activation of hTERT in HCC development
(1) In order to overcome the artificial effect of in vitro overexpression,preS2 antisense RNA were involved.RT-PCR and ELISA showed that preS2 antisense RNA could significantly decrease preS2 mRNA and HBsAg expression. Suppression of preS2 decreased hTERT expression.
(2) 16 clinical tumor samples were involved and grouped by preS2 expression.As expected,much higher hTERT protein expression could be detected in preS2-positive HCC than that of preS2-negative tumors.
(3) Using transfection of preS2 fused with EGFP tag,we verified the nuclei localization of preS2 which is consistent with previous study.This raises the possibility ofpreS2 as a transcriptional activator.
(4) Results of cotransfection and luciferase assay clearly showed the preS2 mediated up-regulation on hTERT promoter activity in different cell lines,including immortal liver cell line LO2,HCC cell lines Be17402 and HepG2,and African green monkey kidney cell line COS-7.The up-regulation in HepG2 cells showed a preS2 dose-dependent manner.These data indicate that preS2 protein can transcriptionally upregulate hTERT via a non- liver-specific manner.
(5) Recent evidence suggests that some virus-encoded proteins contribute to effect of telomerase through the c-myc binding sites.However,we failed to detect any change in c-myc expression after preS2 transfection,which indicated that c-myc play no obvious effect in the preS2-mediated upregulation of hTERT promoter.
(6) To define the region of the hTERT promoter that was necessary for the preS2 response,a serial of hTERT promoter-reporter truncated or deletion constructs (pGL3B-895/pGL3B-371/pGL3B-306/pGL3B-349/pGL3B-329/pGL3B-318/ pGL3B-DELS2/ pGL3B-MUT371R/ pGL3B-MUT349S) were constructed. Cotransfection and luciferase assay demonstrate that preS2 upregulates hTERT promoter via a 65-bp region located in -371 to -307bp.Further assay identified a novel preS2 responsive-region(PRR) located between -349 and -329 bp upstream of the translational start site in the hTERT promoter.
(7) EMSA verified the binding of preS2 to the PRR both in vitro and ex vivo.
2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC
(1) RT-PCR and Western blot results indicate that forced HBV and its viral proteins expression can reduce endogenous ZHX2 expression in SMMC7721.HBV and HBC showed stronger influence compared with HBX and preS2.Inversely, blocking HBV viral proteins expression in HepG2.2.15 can increase ZHX2 expression.Among all genes studies,HBC has the strongest effects.
(2) 33 clinical tumor samples were involved and grouped by HBV infection status. Results showed that much lower ZHX2 mRNA expression could be detected in HBV-positive HCC than that of HBV-negative tumors.(p<0.01)
(3) Studies in mice indicated that ZHX2 has a lower expression level in HBV transgenic mice than that in normal BABLC mice.(p<0.01)
All above results support the hypothesis that HBV could regulate ZHX2 expression in HCC.
3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism
(1) RT-PCR results showed negative correlation between GPC3 and ZHX2 mRNA expression in different human cell lines.Using HepG2 and Hep3B,which express high GPC3 levels,we show that ZHX2 over-expression significantly decreases GPC3 secretion.Furthermore,using LO2 and SMMC7721 cells,which express low GPC3 levels,we use siRNA inhibition to show that GPC3 is de-repressed when ZHX2 levels are reduced.The inverse correlation combined with ZHX2 overexpression and siRNA experiment results indicate that ZHX2 could be responsible for the suppression of GPC3 in human liver cell lines.
(2) 5-Aza-CdR pre-treated HepG2 cells showed an increased expression of ZHX2. Meanwhile,the expression of GPC3 decreased.This also represents the evidence that ZHX2 represses GPC3.
(3) Spearman analysis of ZHX2 and GPC3 protein expression in 33 HCC samples showed an inverse correlation(r=-0.4968,p<0.01).
(4) GPC3 promoter activity was validated in HepG2 and COS-7.Co-transfections of ZHX2 and GPC3-luciferase reporter genes demonstrate ZHX2 repression is governed by the GPC3 promoter.This represents the first direct evidence that ZHX2 represses GPC3.
CONCLUSIONS
1 Anti-sense blocking assay and clinical samples detection assay furtherly validated that HBV preS2 protein can transactivate hTERT.Cotransfection and Dual-luciferase assay proved that preS2 upregulates hTERT expression via the PRR element(-349～-329bp upstream the transcription initial site of hTERT gene) to involve HCC development.
2 Overexpression and anti-sense blocking assay in cell lines,HBV transgene mice model and clinical samples detection assay illuminate that Hepatitis B Virus and its viral proteins suppress ZHX2 expression in HCC.
3 Overexpression/siRNA assay in cell lines and clinical samples detection assay validated that ZHX2 is a repressor of GPC3 expression in HCC.GPC3 core promoter reporter plasmid was constructed successfully.Cotransfection and Dual-luciferase assay showed that ZHX2 effectively suppresses GPC3 promoter activity,which in further validates the suppression role of ZHX2 on GPC3.
INNOVATIONS AND SIGNIFICANCES
1 In this study we have found and defined a novel mechanism of viral liver carcinogenesis:HBV preS2 protein can function as a transactivator,interact with the PRR region of hTERT promoter to regulate telomerase activity,advance the development of HBV related HCC.
2 We also proved that HBV and its viral proteins decreases ZHX2 expression in HCC.These results provide new insight into pathogenic mechanism of HBV related HCC.
3 We have shown that ZHX2 represses AFP and GPC3 expression in human hepatocellular carcinoma.Since both AFP and GPC3 are frequently reactivated in liver tumors and ZHX2 appears to be silenced in some HCC samples,our studies may provide new insight into the development of HCC and may also provide new targets for drug development.
肝细胞肝癌(hepatocellular carcinoma,HCC)是严重危害人类生命的恶性肿瘤,全世界每年新发病例100万,中国是HCC的高发地区,病例数约占全球HCC病例的55%。乙型肝炎病毒(hepatitis B virus,HBV)感染是HCC发生的主要危险因素,临床研究发现63.2%的肝癌患者乙肝表面抗原阳性。因此,揭示HBV相关HCC分子发病机制极为重要和迫切。
HBV相关HCC的发生是多基因、多步骤、多阶段的过程,HBV DNA可以直接整合到宿主细胞基因组发挥其顺式激活作用,也可以通过其编码蛋白发挥反式激活作用。乙肝病毒及其蛋白对宿主多种基因,尤其是肿瘤相关基因的调控,在HCC发生中发挥重要作用。寻找受HBV调控的宿主关键基因并阐明其机制,不仅有助于揭示HBV相关HCC发生机制,还将为HCC治疗提供新靶点。
本研究在前期基础上,结合当今研究进展,选择两种肝癌相关重要宿主基因:端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)及新型转录抑制因子ZHX2(zinc-finger and homeoboxe 2),深入研究HBV对它们的调控作用及其在HCC发生中的作用。
端粒酶的活化和端粒长度的维持与肝细胞永生化密切相关。HBV相关HCC中,100%端粒酶呈激活状态,且端粒酶逆转录酶hTERT表达上调。本室前期研究发现,肝癌细胞系HepG2中过表达HBV反式激活蛋白preS2可以上调hTERT表达,提示preS2介导的hTERT激活是HBV相关HCC发生的重要机制,本研究将在此基础上进一步阐明其机制。
甲胎蛋白(alpha fetoprotein,AFP)是HCC临床检测金指标,在临床上已广泛应用。但该检测还不能完全满足临床要求,所以寻找有助于早期诊断的肝癌特异性标记物具有重要理论意义和临床应用价值。磷脂酰肌醇蛋白聚糖(Glypican3,GPC3)是近年新兴的具有潜力的肝癌标记物。
ZHX2是2003年发现的新型转录抑制因子,广泛存在于脊椎动物体内。目前已知小鼠中受到ZHX2负调控的基因仅有3种,即AFP、H19和GPC3,此三种基因均具有肝癌特异性高表达的特点:在胚胎期高表达,出生后受到抑制,而在肝癌发生时再次被激活。且AFP和GPC3具有癌基因特性,参与HCC的发生。本室前期研究证明ZHX2抑制肝癌细胞系中AFP的表达,提示ZHX2在肝癌发生中具有重要作用。已有报道证明,HCC患者中AFP表达与HBV感染密切相关,但在HCC中HBV是否可以调控ZHX2,以及ZHX2是否调控人肝癌中GPC3的表达,迄今尚未见报道,本研究将对此进行系列研究。
综上,为了深入探讨HBV相关HCC的发病机制,本研究在前期基础上,选择hTERT及ZHX2作为HCC发生的关键基因,拟从以下几个方面进行研究:
1.HBV编码的preS2蛋白反式激活hTERT参与HCC发生的分子机制。
2.HBV对肝癌相关新型转录抑制因子ZHX2的调控及作用研究:
(1)HBV对HCC中ZHX2的调控作用。
(2)ZHX2对HCC中GPC3的抑制作用。
方法
第一部分HBV编码的preS2蛋白反式激活hTERT参与HCC发生的分子机制
1.preS2对hTERT的调控作用
1.1细胞实验:设计合成针对preS2的反义寡核苷酸,脂质体转染HepG2.2.15细胞,RT-PCR、Western blot检测反义封闭preS2后hTERT表达。
1.2临床检测:收集HCC组织标本16例,RT-PCR、Western blot分别检测preS2和hTERT表达。
2.preS2蛋白对hTERT启动子反式激活作用的研究
2.1 preS2-EGFP融合蛋白在肝癌细胞中的表达:构建preS2-EGFP融合蛋白表达载体,脂质体转染HepG2细胞,48h后进行DAPI核染,荧光显微镜观察preS2的表达定位情况。
2.2 preS2对hTERT启动子的激活作用:preS2表达载体(连有HA标签)与hTERT启动子报告质粒共转染肝永生化细胞LO2、肝癌细胞HepG2、BEL7402以及非洲绿猴肾细胞COS-7,双荧光素酶报告基因检测preS2对hTERT启动子的作用;同时在HepG2细胞中以不同剂量preS2表达载体与hTERT启动子报告质粒共转染,验证preS2对hTERT作用的剂量依赖性。
3.preS2反式激活hTERT启动子的分子机制研究
3.1 preS2介导的hTERT启动子激活中c-myc的表达:肝癌细胞HepG2转染preS2表达载体(连有HA标签)后,Western blot检测c-myc蛋白的表达变化。
3.2 preS2激活hTERT启动子关键区段(PRR)的确定:构建系列截短、缺失及突变的hTERT启动子报告质粒,与preS2表达载体共转染HepG2和COS-7,双荧光素酶报告基因检测,确定preS2在hTERT启动子上的作用靶点,命名为preS2-Resiponsible Region,简称PRR。
3.3 EMSA实验:
3.3.1细胞模型:肝癌细胞HepG2转染preS2表达载体后,两步法提取核蛋白。
3.3.2临床HCC组织核蛋白抽提:收集10例preS2阳性和6例阴性新鲜肝癌组织标本,常规抽提核蛋白。
3.3.3 EMSA实验:合成PRR寡核苷酸片段,地高辛标记后作为探针,分别与上述细胞核蛋白和组织核蛋白进行EMSA,检测preS2与PRR的结合。
第二部分HBV对HCC中ZHX2的调控及其作用研究
1.HBV对HCC中ZHX2的调控作用
1.1细胞研究
1.1.1 HBV及其基因片段过表达对HepG2细胞中ZHX2表达的作用:HBV及其编码蛋白HBX、HBC、preS2表达质粒(空载体为对照)脂质体转染HepG2细胞,48h后收集细胞,RT-PCR、Western blot检测ZHX2表达变化。
1.1.2反义抑制HBV不同片段对HepG2.22.15细胞中ZHX2表达的作用:设计合成针对HBX、HBC、preS2的反义寡核苷酸,脂质体转染HepG2.2.15细胞(无关序列转染组为对照),48h后收集细胞,RT-PCR、Western blot检测ZHX2表达变化。
1.2动物实验
6月龄HBV转基因小鼠2只、正常BALB/c对照小鼠3只,取肝组织提取总RNA,实时定量RT-PCR检测两组小鼠肝组织中ZHX2表达差异。
1.3临床检测
收集33例HCC组织标本,按HBV感染情况分为两组,RT-PCR检测两组标本中ZHX2表达差异。
2.ZHX2对HCC中GPC3的抑制作用及其机制的初步探讨
2.1 ZHX2对不同肝癌细胞系中GPC3的抑制作用研究
2.1.1不同肝癌细胞系中ZHX2与GPC3表达的相关性:培养肝癌细胞系HepG2、Hep3B、SMMC7721及肝永生化细胞系LO2,收集对数生长期细胞,RT-PCR检测ZHX2及GPC3的mRNA表达情况。
2.1.2过表达实验:在ZHX2相对低表达的细胞系(HepG2、Hep3B)中,脂质体转染ZHX2表达载体,48h后收集细胞,RT-PCR、Western blot检测GPC3的表达变化。
2.1.3 siRNA基因沉默实验:在ZHX2相对高表达的细胞系(SMMC7721、LO2)中,脂质体转染针对ZHX2的siRNA质粒,干扰ZHX2的表达,48h后收集细胞,RT-PCR检测GPC3的表达变化。
2.1.4去甲基化恢复ZHX2表达对GPC3的影响:不同剂量去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理HepG2细胞恢复ZHX2的表达,RT-PCR、Westernblot检测GPC3的表达变化。
2.2 HCC临床标本中ZHX2与GPC3表达的相关性研究
收集33例HCC组织标本,Western blot检测标本中ZHX2、GPC3表达情况,采用Spearman相关系数分析ZHX2与GPC3表达的关系。
2.3 ZHX2抑制GPC3启动子活性研究
2.3.1 GPC3核心启动子报告质粒载体的构建:设计引物,PCR扩增GPC3转录起始点上游218bp片段,克隆入pGL3-Basic载体,构建GPC3核心启动子荧光素酶报告基因载体pGL3-GPC3-218,分别在HepG2和COS-7细胞中通过基因转染和双荧光素酶报告基因检测验证启动子活性。
2.3.2 ZHX2对GPC3启动子的抑制作用研究:不同剂量ZHX2表达载体与GPC3核心启动子报告质粒共转染HepG2细胞,双荧光素酶报告基因检测ZHX2对GPC3核心启动子的作用。
结果
第一部分HBV编码的preS2蛋白对hTERT反式激活作用的分子机制
1.preS2蛋白对HCC中hTERT表达的调控作用
1.1反义封闭preS2抑制HepG2.2.15细胞中hTERT表达
本室前期研究发现,preS2表达质粒瞬时转染细胞可以明显上调HepG2细胞中hTERT的表达,提示preS2可以增强hTERT表达。为克服基因过表达的人为因素影响,本研究设计合成针对preS2的反义寡核苷酸片段,脂质体转染HepG2.2.15细胞(整合有HBV基因组,可表达HBV蛋白),48h后,收集细胞,RT-PCR、ELISA结果显示反义寡核苷酸可有效降低preS2 mRNA和细胞上清中HBsAg的表达;同时,HepG2.2.15细胞中反义封闭preS2的表达后,hTERT在mRNA和蛋白水平的表达均明显降低。上述结果显示:封闭preS2蛋白可以抑制肝癌细胞系中hTERT表达。
1.2 preS2阳性HCC组织标本中hTERT表达显著高于preS2阴性HCC标本
为进一步研究HCC中preS2对hTERT的调控作用,本研究收集16例HCC组织标本,检测其preS2及hTERT的表达。RT-PCR结果显示:16例HCC标本中,10例HBV阳性的HCC标本其preS2亦呈阳性表达,6例HBV阴性的HCC标本其preS2亦呈阴性表达;Western blot检测显示:10例preS2阳性表达的HCC标本hTERT的表达水平明显高于6例preS2阴性表达的HCC标本(p＜0.05)。上述结果提示,临床HCC标本中preS2表达与hTERT表达正相关,与细胞系中的研究结果相符。
2.preS2蛋白在不同细胞中反式激活hTERT启动子
众多文献证实,hTERT的转录水平调控是端粒酶表达调控的主要方式,为此,本研究构建preS2真核表达载体,研究其对hTERT启动子的调控作用。
2.1 preS2融合蛋白定位表达于肝癌细胞核内
为进一步研究preS2对hTERT的调控作用,本研究成功构建preS2绿色荧光融合蛋白表达载体,命名为pEGFP-preS2;转染HepG2细胞48h后DAPI核染,荧光显微镜观察绿色荧光主要定位于细胞核,显示preS2可在肝癌细胞中表达,并主要表达于细胞核,具有转录因子的特点。
2.2 preS2在不同细胞中反式激活hTERT启动子
pEGFP-preS2与hTERT启动子报告质粒共转染不同细胞系,双荧光素酶报告基因检测结果显示:在肝永生化细胞LO2、肝癌细胞HepG2、BEL7402以及非洲绿猴肾细胞COS-7中,preS2对hTERT启动子均具有明显的反式激活作用,与pcDNA3空载体转染组相比,作用强度分别为2.469倍、2.933倍、2.556倍、2.426倍,提示preS2对hTERT启动子的反式激活作用并非肝细胞特异性;不同剂量preS2表达载体与全长hTERT启动子报告质粒共转染HepG2,双荧光素酶报告基因检测显示:preS2对hTERT启动子反式激活作用具有剂量依赖性。
3.preS2反式激活hTERT启动子分子机制研究
3.1 preS2反式激活hTERT启动子不依赖于c-myc
hTERT启动子区存在2个c-myc结合位点,且文献证实多种病毒通过该位点激活hTERT。然而,本研究中Western blot检测显示:过表达preS2并没有影响c-myc蛋白的表达变化,提示preS2对hTERT启动子的反式激活是不依赖c-myc途径的。
3.2 preS2反式激活hTERT关键区段PRR的确定
为确定preS2激活hTERT的关键片段,本研究成功构建系列截短、缺失及突变的hTERT启动子报告质粒,分别命名为pGL3B-895、pGL3B-371、pGL3B-306、pGL3B-349、pGL3B-329、pGL3B-318、pGL3B-DELS2、pGL3B-MUT371R、pGL3B-MUT349S。上述载体分别与preS2表达载体共转染HepG2和COS-7细胞,双荧光素酶报告基因检测结果分析,将preS2在hTERT启动子上的作用范围确定在-371～-307之间65bp区域,并进一步缩短至-349～-329bp,命名为preS2 responsive-region,简称PRR。
3.3 EMSA分析preS2与PRR的结合
合成PRR寡核苷酸,地高辛标记后与preS2转染的HepG2细胞核蛋白共孵育,凝胶电泳迁移抑制实验(EMSA)结果显示:preS2表达载体转染的核蛋白可与PRR探针结合,此种结合可被特异性PRR片段竞争性抑制,且HA抗体作用后电泳带明显后滞,提示preS2可与hTERT启动子上PRR位点特异性结合。为进一步验证上述结果,收集并抽提HCC组织标本核蛋白,EMSA结果显示:preS2阳性的HCC标本与PRR的结合能力明显强于preS2阴性的HCC标本,与细胞实验结果一致。
第二部分HBV对HCC中ZHX2的调控及其作用研究
ZHX2是2003年发现的新型转录抑制因子,迄今在小鼠中仅发现了三个受ZHX2调控的靶基因:AFP、GPC3及H19。其中,H19为印记基因,而AFP和GPC3均具有肝癌特异性高表达的特点,并参与肝癌发生。本室前期研究发现,ZHX2抑制肝癌细胞系中AFP表达,提示其在肝癌中的重要作用。本部分实验首次探讨HBV对ZHX2的调控作用,并探讨了ZHX2对人肝癌细胞中GPC3的抑制作用,本部分研究将为阐明HBV相关HCC的发生机制提供新的实验依据。
1.HBV对HCC中ZHX2表达的调控作用
本研究中利用细胞模型、转基因动物及临床检测,系统研究HBV对ZHX2的调控作用。
1.1 HBV抑制肝癌细胞系中ZHX2的表达
为研究HBV对ZHX2的抑制作用,分别进行ZHX2过表达实验和反义寡核苷酸抑制实验。RT-PCR、Western blot检测结果显示:肝癌细胞SMMC7721中过表达HBV及其编码蛋白HBX、HBC、preS2后,ZHX2表达下调,其中以HBV和HBC的影响更为明显;HepG2.2.15细胞中反义封闭HBX、HBC、preS2表达后,ZHX2表达上调,其中封闭HBC表达后ZHX2表达上调更为明显。上述结果提示,HBV及其基因片段可以抑制肝癌细胞中ZHX2表达,且HBC抑制作用最强。
1.2 HBV转基因鼠肝组织中ZHX2表达显著低于对照鼠
HBV转基因鼠肝组织稳定表达HBV及其抗原,是研究HBV感染的良好动物模型。本研究选择6月龄HBV转基因小鼠,并与正常同系BALB/c小鼠为对照,抽提肝组织RNA,实时定量RT-PCR检测ZHX2表达。结果显示:HBV转基因小鼠肝组织中ZHX2表达明显低于正常对照组。(p＜0.001)
1.3 HBV阳性HCC标本中ZHX2 mRNA显著低于HBV阴性组
为进一步验证HBV对ZHX2的抑制作用,收集33例HCC组织标本,RT-PCR检测ZHX2表达,结果显示:HBV阳性组标本中ZHX2 mRNA的表达水平明显低于HBV阴性组。(p＜0.01),与预期一致。
2.ZHX2对HCC中GPC3的抑制作用及其机制的初步探讨
2.1 ZHX2抑制人肝癌细胞系中GPC3的表达
RT-PCR结果显示,四种肝癌细胞系中ZHX2与GPC3表达负相关:在ZHX2相对低表达的细胞系HepG2、Hep3B中GPC3相对高表达;在ZHX2相对高表达的细胞系SMMC7721、LO2中GPC3相对低表达。
ZHX2转染实验结果表明,HepG2、Hep3B细胞系中过表达ZHX2后,GPC3的表达水平降低;siRNA基因沉默实验证明,在SMMC7721、LO2细胞系中干扰ZHX2的表达后,GPC3的表达水平显著升高。
有研究显示,甲基化可以抑制ZHX2表达,为进一步研究ZHX2对GPC3的调控作用,利用不同浓度去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理HepG2细胞(ZHX2低表达)。RT-PCR、Western blot检测结果显示:去甲基化可以升高ZHX2的表达水平;且随着去甲基化药物浓度的升高,GPC3的表达降低。
上述结果均提示,ZHX2可以抑制人肝癌细胞系中GPC3的表达。
2.2 HCC组织标本中ZHX2表达与GPC3负相关
为进一步验证HCC标本中ZHX2对GPC3的调控作用,收集33例HCC组织标本,Western blot检测ZHX2和GPC3表达后,半定量分析其表达水平,并进行Spearman相关系数分析,结果显示:ZHX2与GPC3呈中度负性相关关系且具有统计学意义(r=-0.4968,p＜0.01)。
2.3 ZHX2明显抑制GPC3核心启动子活性
2.3.1 GPC3核心启动子荧光素酶报告基因载体的构建
PCR扩增GPC3基因转录起始点上游218bp片段,克隆入pGL3-Basic质粒,构建GPC3核心启动子荧光素酶报告基因载体。基因转染实验及双荧光素酶报告基因检测结果显示:所构建的GPC3核心启动子报告质粒在HepG2和COS-7细胞中均具有启动子活性。
2.3.2 ZHX2抑制GPC3核心启动子活性
ZHX2表达载体与GPC3核心启动子报告质粒共转染HepG2细胞,双荧光素酶报告基因检测结果显示:ZHX2对GPC3核心启动子具有明显抑制作用(p＜0.01)。
结论
1.细胞系反义封闭实验和临床标本检测进一步验证HBV编码的preS2蛋白对hTERT的激活作用。共转染双荧光素酶报告基因检测和EMSA实验证实preS2蛋白通过作用于hTERT启动子的PRR区域(位于hTERT转录起始点上游-349～-329bp),反式激活hTERT进而活化端粒酶,从而参与HCC的形成。
2.从细胞系过表达和反义封闭实验、HBV转基因小鼠动物模型以及临床标本检测分析三方面证实:在HCC中,HBV可以抑制新型转录抑制因子ZHX2的表达,进而参与HCC发生和发展。
3.细胞系过表达和小干扰实验以及临床标本检测分析证实:ZHX2抑制HCC中GPC3的表达;成功构建人GPC3基因核心启动子报告质粒,共转染及双荧光素酶报告基因检测显示ZHX2可有效抑制GPC3启动子活性,在转录水平进一步验证了ZHX2对GPC3的表达抑制作用。
创新性和意义
1.本研究提出HBV编码的preS2蛋白通过调控端粒酶活性,进而参与细胞恶性变的论点,为阐明HBV相关肝细胞肝癌致病机制提供新思路。发现preS2反式激活hTERT的关键靶点,为HBV活化端粒酶提供直接证据。
2.众多研究提示ZHX2是HCC中一种潜在的抑癌基因。本研究首次证实HBV在HCC发生中抑制ZHX2的表达,为阐明HBV相关HCC的发病机制提供新的思路。
3.本研究首次证实在HCC中ZHX2对GPC3的抑制作用,并报道人ZHX2对GPC3启动子的抑制作用,为进一步探讨ZHX2对GPC3抑制作用的分子机制以及发现新的肝癌标志物奠定基础。
OBJECTIVES
Hepatocellular carcinoma(HCC) is a kind of malignant tumor that affects human health severely.China is HCC high-incidence district and takes up 55%of HCC cases in the world.HCC incidence in developed countries is increasing in recent years. Chronic hepatitis B virus(HBV) infection has been identified as a leading risk factor for HCC in China.So it is urgent to reveal the mechanisms of HBV related HCC.
The mechanism of HBV related HCC is complicated.The integration of HBV-DNA into the host genome can trigger cis-activation directly;Also regulatory proteins HBX and preS2 activators encoded by integrated subviral HBV genomes can exert a tumor promoter-like function,resulting in positive selection of cells producing a functional regulatory protein.To search the key genes regulated by HBV,not only provides newsights into mechanisms of HBV related HCC development,but also gives new potential targets for drug design.
Here,two important HCC related genes,human telomerase reverse transcriptase (hTERT) and zinc-finger and homeoboxe 2(ZHX2) were selected to be further studied.
It has been proved that telomerase activation is an important event during the process of immortalization and upregulation of hTERT might be a critical event in the development of human cancers,including HCC.Earlier studies from our laboratory have shown that preS2 overexpression can upregulate both hTERT expression and telomerase activity in HepG2 cells,resulting in enhanced cell growth which could be abolished by knocking down of hTERT in the cell,indicating that preS2 involves in HCC via hTERT upregulation.However,what's the mechanism remains unknown.In the first part of the study,cotransfection and report assay were involved to explore the molecular mechanism of preS2-mediated hTERT upregulation.
Zinc-finger and homeoboxes 2(ZHX2) gene belongs to a small family of transcription factors that also includes ZHX1 and ZHX3.All ZHX proteins contain two C2-H2 zinc-finger motifs and four homeodomains,are ubiquitously expressed and localized to the nuclei of cells,and appear to function as transcriptional repressors. Insight into the biological function of ZHX2 has come from the analysis of alpha-fetoprotein(AFP) expression in BALB/cJ mice.ZHX2 is reported to be responsible for adult AFP repression in BALB/cJ mice.H19 and Glypican 3 are two additional targets of ZHX2 in the mice liver and have the same expression law as AFP. Our previous studies show that ZHX2 is a repressor of AFP in HCC cell lines,which indicates the potential role of ZHX2 in HCC development.In the second part of this study,we aim to explore if HBV regulate ZHX2 and if ZHX2 is responsible for GPC3 regulation in HCC.
In order to investigate the mechanisms of HBV related HCC and gene regulation during HCC development,we focus our studies on the following aspects:
1.The precise mechanism for preS2 mediated transcriptional activation of the hTERT gene in HCC development.
2.The role of ZHX2 in HBV related HCC:
(1) The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC.
(2) The regulation of ZHX2 on GPC3 expression in HCC and the mechanism.
METHODS
1.The molecular mechanism for preS2 mediated transcriptional activation of the hTERT in HCC development
(1) HepG2.2.15 cells were pre-treated with preS2 antisense RNA to block the expression of preS2,RT-PCR and Western blot were applied to detect the expression change of hTERT.
(2) 16 HCC samples were collected,RT-PCR and Western blot were applied to detect the expression of preS2 and hTERT respectively.
(3) preS2-EGFP fusion protein eukaryotic expression vector was constructed and named pEGFP-preS2.48h After transfected into HepG2 cells,the cells were stained with 4',6-diamidino-2-phenylindole(DAPI).Digital images were taken from a OlympusⅨ81 fluorescence microscope with 400×magnification.
(4) 0.5μg hTERT luciferase reporter and 0.5μg preS2 expression plasmids were cotransfected into HepG2/BEL7402/LO2/COS-7.Meanwhile,different dose preS2 expression plasmids were used in HepG2.20ng SV40-Renilla was used as control to standardize the transcription efficiency.Cells were harvested 48h later in reporter lysis buffer.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocol.
(5) HepG2 cells were transfected with pcS2-HA expression plasmids(preS2 gene fused with hemaglutinin tag) or pcDNA3.Western blot was performed to detect the expression change of c-myc.
(6) To define the region of the hTERT promoter that was necessary for the preS2 response,series truncated/deleted/mutated hTERT promoter reporter plasmids were constructed.Luciferase assays were performed after cotransfected with pcS2-HA in HepG2 and COS-7.
(7) In order to further explore if preS2 could bind with PRR,EMSA were involved with nuclear extract from both HepG2 cells transfected with pcS2-HA and freshly isolated HCC samples.
2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC
(1) HBV and its viral proteins HBX/HBC/preS2 were overexpressed in HepG2 cells. Antisense RNA against HBX/ HBC/ preS2 were involved in HepG2.2.15. RT-PCR and Western blot were applied to detect the expression change of ZHX2.
(2) 33 HCC samples were grouped by HBV infection status,RT-PCR was applied to detect the expression difference of ZHX2;HBV DNA loads were detected by real-time quantitative PCR with the HBV-positive HCC tissue genomic DNA, ZHX2 expression levels were analyzed with HBV DNA.
(3) Total RNA of the liver tissues were extracted by TRIZOL from 2 HBV transgene mice and 3 normal BALB/c.All the mice were 6 months old.ZHX2 expression levels were determined by real-time quantitative PCR.
3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism
(1) RT-PCR was applied to detect the different expression levels of ZHX2 and GPC3 in HCC cell lines HepG2、Hep3B、SMMC7721 and immortalized liver cell line LO2.ZHX2 was overexpressed in cell lines express low ZHX2 levels.In contrast, siRNA-mediated ZHX2 knockdown was applied in cell lines express high ZHX2 levels.Then RT-PCR and Western blot were applied to detect the expression change of GPC3.
(2) HepG2 cells were treated with 5-Aza-CdR of different dose to recover ZHX2 expression.RT-PCR and Western blot were applied to detect the expression change of GPC3.
(3) 33 HCC samples were collected.ZHX2 and GPC3 expression were detected by Western blot.The relationship of ZHX2 and GPC3 expression was analyzed by Spearman analysis.
(4) GPC3 core promoter reporter plasmid was constructed and named pGL3-GPC3-218.The promoter activity was validated in HepG2 and COS-7 by Dual-Luciferase Reporter Assay.Different dose of pcDNA3-ZHX2-HA(ZHX2 expression vector with HA tag) were cotransfected with pGL3-GPC3-218. pRL-TK plasmids were involved in each group to normalize transfection efficiency.Cells were harvested 48h later for luciferase assay.
RESULTS
1.The molecular mechanism for preS2-mediated transcriptional activation of hTERT in HCC development
(1) In order to overcome the artificial effect of in vitro overexpression,preS2 antisense RNA were involved.RT-PCR and ELISA showed that preS2 antisense RNA could significantly decrease preS2 mRNA and HBsAg expression. Suppression of preS2 decreased hTERT expression.
(2) 16 clinical tumor samples were involved and grouped by preS2 expression.As expected,much higher hTERT protein expression could be detected in preS2-positive HCC than that of preS2-negative tumors.
(3) Using transfection of preS2 fused with EGFP tag,we verified the nuclei localization of preS2 which is consistent with previous study.This raises the possibility ofpreS2 as a transcriptional activator.
(4) Results of cotransfection and luciferase assay clearly showed the preS2 mediated up-regulation on hTERT promoter activity in different cell lines,including immortal liver cell line LO2,HCC cell lines Be17402 and HepG2,and African green monkey kidney cell line COS-7.The up-regulation in HepG2 cells showed a preS2 dose-dependent manner.These data indicate that preS2 protein can transcriptionally upregulate hTERT via a non- liver-specific manner.
(5) Recent evidence suggests that some virus-encoded proteins contribute to effect of telomerase through the c-myc binding sites.However,we failed to detect any change in c-myc expression after preS2 transfection,which indicated that c-myc play no obvious effect in the preS2-mediated upregulation of hTERT promoter.
(6) To define the region of the hTERT promoter that was necessary for the preS2 response,a serial of hTERT promoter-reporter truncated or deletion constructs (pGL3B-895/pGL3B-371/pGL3B-306/pGL3B-349/pGL3B-329/pGL3B-318/ pGL3B-DELS2/ pGL3B-MUT371R/ pGL3B-MUT349S) were constructed. Cotransfection and luciferase assay demonstrate that preS2 upregulates hTERT promoter via a 65-bp region located in -371 to -307bp.Further assay identified a novel preS2 responsive-region(PRR) located between -349 and -329 bp upstream of the translational start site in the hTERT promoter.
(7) EMSA verified the binding of preS2 to the PRR both in vitro and ex vivo.
2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC
(1) RT-PCR and Western blot results indicate that forced HBV and its viral proteins expression can reduce endogenous ZHX2 expression in SMMC7721.HBV and HBC showed stronger influence compared with HBX and preS2.Inversely, blocking HBV viral proteins expression in HepG2.2.15 can increase ZHX2 expression.Among all genes studies,HBC has the strongest effects.
(2) 33 clinical tumor samples were involved and grouped by HBV infection status. Results showed that much lower ZHX2 mRNA expression could be detected in HBV-positive HCC than that of HBV-negative tumors.(p<0.01)
(3) Studies in mice indicated that ZHX2 has a lower expression level in HBV transgenic mice than that in normal BABLC mice.(p<0.01)
All above results support the hypothesis that HBV could regulate ZHX2 expression in HCC.
3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism
(1) RT-PCR results showed negative correlation between GPC3 and ZHX2 mRNA expression in different human cell lines.Using HepG2 and Hep3B,which express high GPC3 levels,we show that ZHX2 over-expression significantly decreases GPC3 secretion.Furthermore,using LO2 and SMMC7721 cells,which express low GPC3 levels,we use siRNA inhibition to show that GPC3 is de-repressed when ZHX2 levels are reduced.The inverse correlation combined with ZHX2 overexpression and siRNA experiment results indicate that ZHX2 could be responsible for the suppression of GPC3 in human liver cell lines.
(2) 5-Aza-CdR pre-treated HepG2 cells showed an increased expression of ZHX2. Meanwhile,the expression of GPC3 decreased.This also represents the evidence that ZHX2 represses GPC3.
(3) Spearman analysis of ZHX2 and GPC3 protein expression in 33 HCC samples showed an inverse correlation(r=-0.4968,p<0.01).
(4) GPC3 promoter activity was validated in HepG2 and COS-7.Co-transfections of ZHX2 and GPC3-luciferase reporter genes demonstrate ZHX2 repression is governed by the GPC3 promoter.This represents the first direct evidence that ZHX2 represses GPC3.
CONCLUSIONS
1 Anti-sense blocking assay and clinical samples detection assay furtherly validated that HBV preS2 protein can transactivate hTERT.Cotransfection and Dual-luciferase assay proved that preS2 upregulates hTERT expression via the PRR element(-349～-329bp upstream the transcription initial site of hTERT gene) to involve HCC development.
2 Overexpression and anti-sense blocking assay in cell lines,HBV transgene mice model and clinical samples detection assay illuminate that Hepatitis B Virus and its viral proteins suppress ZHX2 expression in HCC.
3 Overexpression/siRNA assay in cell lines and clinical samples detection assay validated that ZHX2 is a repressor of GPC3 expression in HCC.GPC3 core promoter reporter plasmid was constructed successfully.Cotransfection and Dual-luciferase assay showed that ZHX2 effectively suppresses GPC3 promoter activity,which in further validates the suppression role of ZHX2 on GPC3.
INNOVATIONS AND SIGNIFICANCES
1 In this study we have found and defined a novel mechanism of viral liver carcinogenesis:HBV preS2 protein can function as a transactivator,interact with the PRR region of hTERT promoter to regulate telomerase activity,advance the development of HBV related HCC.
2 We also proved that HBV and its viral proteins decreases ZHX2 expression in HCC.These results provide new insight into pathogenic mechanism of HBV related HCC.
3 We have shown that ZHX2 represses AFP and GPC3 expression in human hepatocellular carcinoma.Since both AFP and GPC3 are frequently reactivated in liver tumors and ZHX2 appears to be silenced in some HCC samples,our studies may provide new insight into the development of HCC and may also provide new targets for drug development.
引文
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