HBV编码的反式激活蛋白调控端粒酶活性的作用及机制研究鼠α白蛋白表达调控机制的研究
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摘要
第一部分HBV编码的反式激活蛋白调控端粒酶活性的作用及机
乙型肝炎病毒(Hepatitis B Virus,HBV)感染肝细胞所致急慢性肝炎及相关肝硬化、肝细胞肝癌(Hepatocellular Carcinoma,HCC)是危害全世界,尤其是我国人民身心健康的重要病因。深入阐明HBV致癌机制,必将为预防和控制肝癌的发生发展提供强有力的手段。目前HBV致癌的确切机理尚未完全清楚,但HBVDNA整合及整合后通过反式激活作用致癌的论点已成为大多数学者的共识。有研究表明在不同的HBV相关HCC组织中,端粒酶编码基因内存在HBV基因片段的整合。这些整合的HBV基因可激活人端粒酶逆转录酶(human telomerasereverse tramscriptase,hTERT),上调端粒酶活性,促进肝癌的发生。
端粒酶(telomerase)位于端粒末端,是一种特殊的核糖核蛋白逆转录酶,大多数正常体细胞不表达端粒酶活性,而在恶性肿瘤中端粒酶活性高表达。研究表明一些病毒编码的蛋白,可通过激活端粒酶来维持端粒长度,阻止细胞死亡,来促进细胞的转化与肿瘤的发生。
HBx与PreS2作为HBV基因编码的两种重要转录激活蛋白,在HBV相关肝癌中的整合率远大于其他蛋白,被认为在HBV相关肝癌的发生发展中起着非常重要的作用。而这两种HBV基因编码的重要转录激活因子,能否上调端粒酶活性,反式激活hTERT启动子促进hTERT转录,以及通过哪些元件激活hTERT启动子,目前还尚未有相关报道。因此,在前期工作的基础上,本课题在国内外率先开展了有关HBV编码反式激活蛋白调控hTERT转录水平的作用及机制研究,为进一步阐明HBV感染相关疾病的发病机制提供新的理论依据。
目的:
1.研究HBx与PreS2蛋白能否促进端粒酶逆转录酶表达,上调端粒酶活性。
2.研究HBx促进端粒酶逆转录酶基因表达的机制,筛选HBx作用于hTERT启动子上关键位点
3.筛选调控hTERT启动子的HBx关键区段
方法:
1.HBx及PreS2蛋白对端粒酶活性及hTERT基因表达的影响
1.1过表达HBx及PreS2蛋白对肝癌细胞端粒酶活性的影响
提取pcDNA3-HBx及空载体对照pcDNA3瞬时转染细胞与未转染对照组HepG2细胞总蛋白;提取稳定表达preS2的HepG2肝癌细胞总蛋白;设立293细胞为端粒酶活性阳性对照组。85℃加热10分钟,灭活293细胞蛋白中端粒酶活性,设为端粒酶活性阴性对照。TRAP法检测端粒酶活性。
1.2过表达HBx及PreS2蛋白对肝癌细胞hTERT基因mRNA水平表达的影响
半定量RT-PCR分别检测pcDNA3-HBx、pcDNA3瞬时转染及未转染HepG2细胞中hTERT mRNA的表达。
半定量RT-PCR分别检测稳定表达preS2的HepG2细胞、pcDNA3空载体对照组细胞及未转染HepG2细胞中hTERT mRNA的表达。
1.3反义封闭HBx与PreS2基因表达对hTERT基因mRNA水平的调控作用
设计合成针对HBx、PreS2的硫代反义寡核苷酸PS-asODNs/HBx、PS-asODNs/preS2及无关对照硫代寡核苷酸PS-asRandom,并以阳离子脂质体介导的方式分别瞬时转染HepG2.2.15细胞系,作用48h后,半定量RT-PCR分别检测各反义寡核苷酸作用组及空细胞对照组hTERT mRNA的表达。
2.HBx对hTERT启动子的调控作用
2.1系列缺失及突变hTERT启动子报告载体的构建
设计、合成针对hTERT启动子不同片段及突变序列的特异性引物,以pCR-TRTP为模板,PCR扩增获得hTERT启动子系列缺失及突变序列的基因。PCR反应产物经酶切定向插入报告基因载体pGL3-basic内,转化大肠杆菌感受态细胞JM109,经抗性筛选、酶切、PCR、DNA测序鉴定获得阳性重组子。
2.2 HBx对全长hTERT启动子的调控作用
将不同剂量的重组子pcDNA3-HBx与携带有hTERT启动子的报告质粒pGL3B-TRTP共转染不同细胞系,同时转染pRL-tk作为内参照。pGL3B-TRTP可在hTERT启动子的作用下表达萤火虫荧光素酶,而pRL-tk可表达海肾荧光素酶。36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析,以均值±标准差表示。
2.3 HBx作用于hTERT启动子关键区域的寻找
将HBx表达载体与系列缺失hTERT启动子报告载体共转染,36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析。
2.4 HBx作用于hTERT启动子关键位点的寻找
将HBx表达载体与系列突变hTERT启动子报告载体共转染,36h后,收集并裂解细胞,测定双荧光素酶表达量,结果进行统计学分析。
2.5 HBx转染前后对Sp1表达水平的影响
提取pcDNA-HBx及pcDNA3瞬时转染与未转染HepG2细胞的各组细胞总蛋白及总RNA,Western blot及RT-PCR检测各组细胞中Sp1,HBx和β-actin的表达水平。
3.调控hTERT启动子的最小HBx关键区域的寻找
3.1截短型HBx重组子的构建
设计、合成针对HBx不同片段基因序列的特异性引物,以pcDNA3-HBx为模板,PCR扩增获得HBx不同截短片段的基因。PCR反应产物经酶切定向克隆入双酶切的pcDNA3-HBx载体内,转化大肠杆菌感受态细胞DH5α,经抗性筛选、酶切、PCR及DNA测序鉴定获得阳性重组子。
3.2截短型HBx在细胞中的表达
系列截短型HBx重组子经脂质体包裹转染HepG2细胞系,48小时后,抽提各组细胞总蛋白与总RNA,Western blot或RT-PCR检测截短型HBx的表达情况。
3.3系列截短型HBx对端粒酶启动子的作用
将系列截短型HBx重组子与携带有hTERT启动子的报告质粒pGL3B-TRTP共转染肝癌细胞系HepG2,同时转染pRL-tk作为内参照。36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析。
结果
1.HBx及PreS2蛋白上调hTERT mRNA表达,促进端粒酶活性
1.1过表达HBx及PreS2蛋白上调端粒酶活性
TRAP实验显示,在各组细胞中均检测到36bp的内参条带,且条带亮度均一。这说明PCR过程中,Taq酶活性正常,上样量一致。阳性对照组293细胞出现相差6bp的DNA梯形条带,而在阴性对照组,未见梯形条带。稳定或瞬时转染有PreS2或HBx的细胞中DNA梯形条带增多,强度增强,说明HBx及PreS2蛋白过表达可上调端粒酶活性。
1.2过表达HBx及PreS2蛋白促进hTERT基因mRNA表达
半定量RT-PCR结果显示,表达有PreS2或HBx的细胞和对照组细胞相比,hTERT基因mRNA水平明显上调(P＜0.05)。
1.3反义封闭HBx与PreS2基因下调hTERT基因mRNA表达
半定量RT-PCR结果显示,反义寡核苷酸PS-asODNs/HBx、PS-asODNs/preS2能分别有效抑制HBx与PreS2基因的表达。在不影响β-actin基因片段表达的情况下,反义封闭HBx与PreS2基因均可以下调HepG2.2.15细胞中hTERT mRNA的表达。
2.HBx通过Sp1结合位点发挥激活hTERT启动子作用
2.1成功构建系列缺失与突变型hTERT启动子报告载体
PCR成功扩增系列缺失与突变型hTERT启动子基因片段,酶切、连接构建重组报告载体,并成功转化宿主菌,经抗性筛选、酶切鉴定、DNA测序分析获得阳性重组报告载体。
2.2 HBx可反式激活全长hTERT启动子
共转染及双荧光检测系统显示,重组子pCDNA3-HBx作用下的pGL3B-TRTP荧光素酶表达强度较空质粒pcDNA3作用组明显升高,约1.5倍左右(p＜0.05)。HBx对pGL3B-TRTP的激活作用具有剂量依赖性,Western-blot检测显示,随着转染pCDNA3-HBx表达载体剂量的增加,HBx蛋白表达增加,Actin表达没有明显变化,但双荧光检测系统显示荧光素酶表达强度增加。pcDNA3-HBx对pGL3B-TRTP激活作用同样可以在其他三种细胞系Bel7402,SMMC-7721,Cos-7中检测到,说明HBx可以反式激活不同细胞中的hTERT启动子。
2.3 HBx反式激活hTERT的关键区段是启动子-197～+37bp
共转染及双荧光检测系统显示,重组子pCDNA3-HBx作用下的pGL3B-TRTP,pGL3B-895,pGL3B-445,pGL3B-371,pGL3B-306,pGL3B-197报告质粒,其相对荧光素酶表达强度比pcDNA3作用下的表达强度分别增加1.45倍,1.40倍,1.46倍,1.38倍,1.58倍与1.38倍。而hTERT启动子5'端缺失到转录起始点下游37bp时,HBx诱导的荧光素酶表达强度消失。这说明HBx通过hTERT启动子-197～+37区域促进启动子转录。
2.4 HBx通过hTERT启动子上Sp1结合位点发挥激活作用
-197～+37区域内存在5个Sp1结合位点和2个C-myc结合位点。为研究Sp1及C-myc在HBx反式激活hTERT启动子中的作用,构建Sp1结合位点、C-myc结合位点突变的hTERT启动子系列突变报告质粒,并分别与HBx表达载体共转染,结果显示,当单独或联合突变C-myc结合位点时,HBx仍可以促进hTERT启动子作用下的荧光素酶表达强度,说明HBx不是通过C-myc结合位点发挥反式激活作用。当突变了Sp1结合位点2,4,5位时,HBx作用于hTERT启动子的荧光素酶表达强度减弱,与pcDNA3作用组没有明显差异(P＞0.05),说明Sp1结合位点2,4,5位在HBx反式激活hTERT启动子活性中发挥重要作用。
2.5 HBx没有改变Sp1表达水平
Western blot结果,经光密度软件分析,计算Sp1蛋白水平的相对表达强度,结果显示,pcDNA3-HBx转染组与pcDNA3转染组及未转染HepG2细胞组相比,Spl蛋白相对表达强度无显著性差异(P＞0.05)。
半定量RT-PCR结果显示,HBx转染后的HepG2细胞,Sp1 mRNA表达水平与未转染HepG2细胞相比无显著差异(P＞0.05)。
3.激活hTERT启动子的最小HBx基因片段为50-140aa
3.1成功构建系列截短型HBx表达载体
PCR成功扩增系列截短型HBx基因片段,酶切、连接构建重组表达载体,并成功转化宿主菌,经抗性筛选、酶切、PCR、DNA测序分析获得阳性重组子。
3.2系列截短型HBx可在细胞内表达
RT-PCR结果显示,转染有不同截短型HBx肝癌细胞内,可扩增出对应分子量的DNA条带,而在转染pcDNA3的HepG2细胞中没有特异性扩增产物。Western-blot结果显示,转染有不同截短型HBx肝癌细胞内,均见较强的actin蛋白表达,而系列截短型HBx仅检测到TC-2、TC-3、HBX、TCN和TN-1在肝癌细胞HepG2中的表达,未检测到TC-1与TN-2在肝癌细胞中的蛋白表达,这与TC-1、TN-2蛋白分子量较小有关。在转染pcDNA3的HepG2细胞中没有检测到HBx截短型蛋白的表达。
3.3系列截短型HBx对端粒酶启动子的作用
共转染及双荧光检测系统显示,当羧基端缺失14个氨基酸时,即TC-3,HBx仍具有反式激活hTERT启动子作用,当缺失24个氨基酸时,甚或缺失104个氨基酸时,其反式激活作用消失。当氨基端缺失50个氨基酸时,即TN-1,截短HBx仍具有反式激活hTERT启动子作用,但缺失97个氨基酸时,其作用消失。同时在构建的TCN重组载体中也验证了当缺失氨基端前50个氨基酸及羧基端14个氨基酸时,截短型HBx仍具有激活hTERT启动子作用,这说明HBx反式激活hTERT启动子的关键区段在50-140氨基酸区域内。
结论
1.通过过表达及反义封闭实验,证实HBx及PreS2蛋白均可以促进肝癌细胞中hTERT mRNA表达,上调端粒酶活性。
2.HBx在不同细胞系以剂量依赖的方式激活hTERT启动子活性。
3.HBx通过hTERT启动子-197～+37激活其转录表达。
4.HBx通过Sp1结合位点,而不是C-myc结合位点发挥激活hTERT启动子的作用。
5.研究不同的截短型HBx对hTERT启动子的作用,初步找到激活hTERT启动子的HBx关键区段在50～140aa区域内。
意义
该课题的完成不仅为HBV相关HCC机制的阐明提供了新的实验依据,为端粒酶调控研究提供新思路,并为HBV相关HCC的治疗提供新的靶点,具有良好的应用前景。
α白蛋白(a-albumin,又名afamin，简称AFM)是第四个被鉴定的白蛋白家族成员，该家族成员还包含其他三种蛋白：白蛋白(albumin，简称Alb)，甲胎蛋白(a-fetoprotein,AFP)，以及维他命D结合蛋白(Vitamin D-binding protein，DBP)。白蛋白家族成员的编码基因均位于人类第四对染色体的4q11．q22区域或小鼠第十四对染色体上，编码蛋白具有相同的框架结构，在转运血清蛋白中发挥重要的作用，主要在肝组织中表达。但这四个家族成员在肝脏的发育过程有不同的表达形式，因而白蛋白家族是研究肝脏特异性基因表达调控的重要模型。有研究表明AFP及AFM参与肝癌的发生。目前众多国内外学者已经详细研究了如何在转录水平调控Alb、AFP与DBP基因的表达，而关于如何调控AFM基因的表达，研究尚少。
该课题在前期研究的基础上，率先分析了AFM基因启动子活性调控区域，为揭示α白蛋白在肝脏发育过程的调控机制及肝癌发生机制提供新的思路。
目的：
1．分析AFM基因启动子活性区域
2．寻找并研究调控AFM基因转录的调控因子
方法：
1．AFM基因启动子报告质粒的构建与活性分析
分别用双酶切和PCR方法从载体mAFM-LacZ上获得全长与系列缺失或突变的小鼠AFM基因启动子序列，分别克隆到pGL3-basic多克隆位点，构建含有AFM启动子的报告质粒。重组子经酶切、PCR及测序鉴定。将重组子分别转染不同的细胞系Hep3B，HepG2及HEK293细胞，通过检测双荧光素酶表达强度，评估不同的报告质粒中AFM启动子活性。
2．转录因子HNFl对AFM启动子的调控作用
将不同剂量的HNFl真核表达质粒pcDNA3-HNFlQ或pcDNA3-HNFlB单独与AFM启动子报告质粒pGL3-mAFM共转染HEK293细胞，同时转染pRL-CMV作为内参照，检测双荧光素酶表达强度，结果进行统计学分析，以均值±标准差表示。
3．转录因子HNF1对突变型AFM启动子的调控作用
将0．1μg的HNF1真核表达质粒peDNA3-HNF1α或pcDNA3-HNF1β单独与不同突变型AFM启动子报告质粒共转染HEK293细胞，检测双荧光素酶表达强度，结果进行统计学分析。
4．转录因子HNF1α与HNF1β联合对AFM启动子的调控作用
将不同剂量的HNF1β表达载体pcDNA3-HNF1β与0．1μg的HNF1α表达载体pcDNA3-HNFl 0【联合与AFM启动子报告质粒pGL3-mAFM共转染HEK293细胞，检测双荧光素酶表达强度，结果进行统计学分析。
5．EMSA分析转录因子HNFI与AFM启动子相互作用研究
针对TESS软件分析得到的AFM启动子上两个HNFl结合位点site1及site2(-145及-75处)，设计合成分别携带野生型和突变型HNF1反应元件的探针，利用Hep3B与转染有pcDNA3-HNF1α或peDNA3-HNF1β质粒的HEK293细胞核提取物，EMSA分析转录因子HNFl与AFM启动子序列结合情况。
6．转录因子C／EBP对AFM启动子的调控作用
将C／EBP真核表达质粒pcDNA3-CEBPa或／和pcDNA3-CEBPp单独或联合与AFM启动子报告质粒pGL3-mAFM共转染Hep3B细胞，检测双荧光素酶表达强度。
结果：
1．成功构建AFM基因启动子报告质粒
经测序证明成功构建了小鼠系列缺失与突变AFM启动子的报告基因质粒。共转染及双荧光素酶检测系统显示，pGL3-mAFM在Hep3B与HepG2中荧光表达量分别为54．91±3．60与6．70±o．08，而在人胚肾细胞系HEK293中所构建重组子荧光素表达极低2．43±0．17，说明所构建的载体pGL3-mAFM具有特异性的AFM启动子活性。缺失型报告载体pGL3-248与pGL3．158的荧光素酶随着缺失序列的增加，表达强度略有减少，但当缺失了从-158～-138之间的序列，pGL3-138荧光素酶相对表达强度明显下降，下降约90％左右；突变载体pGL3-HNF l mtl荧光素酶表达强度同样下降90％左右。突变载体pGL3-HNF1mt2荧光素酶表达强度下降50％左右，当联合突变HNF1两个结合序列时，pGL3-HNF1mt12荧光素酶强度基本消失，说明转录起始点上游336bp序列是AFM启动子核心序列，HNF-1结合位点是维持AFM启动子活性重要元件。
2．转录因子NNF1对AFM启动子的激活作用
HNF1表达载体pcDNA3-HNF1α或pcDNA3-HNF1β单独与PGL3-mAFM报告载体共转染，随着HNF1α或HNF1β剂量的增加，AFM启动子作用下的荧光素酶表达强度逐渐增加，但HNF1β的激活作用仅为HNF1α作用的10％左右。
3．转录因子HNF1对突变型AFM启动子的激活作用
HNF1表达载体pcDNA3-HNF1α或pcDNA3-HNF1β单独作用于突变型AFM表达载体时，结果显示，当单独突变sitel或site2结合位点时，HNFla作用下的转录活性相比野生型AFM启动子组分别下降50％与40％左右；当单独突变sffel结合位点时，HNF1β作用下的突变型启动子转录活性较野生型AFM转录活性未见改变，而突变site2结合位点时，HNF1β作用下的转录活性较野生型AFM下降50％。说明HNF1α转录因子可通过Site1及site2发挥激活启动子作用，而HNF1β主要通过site2发挥激活AFM启动子作用。当同时突变sitel及site2位点时，HNF1α或HNF1β作用下的转录活性明显下降，与pGL3-basic相比，HNF1α或HNF1β对双突变pGL3-HNF1mt12仍有弱的激活作用，提示在AFM启动子区域存在其他弱的HNF1结合位点。
4．转录因子HNF1β抑制HNF1α激活AFM启动子作用
转录因子HNF1α与HNF1β表达载体与AFM启动子共转染，结果显示随着HNF1β剂量的增加，HNF1α作用下的AFM转录活性逐渐下降。
5．AFM基因启动子区HNF1反应元件与蛋白相互作用分析
EMSA结果显示，AFM启动子上两个HNF1结合位点(site1与site2)都可与核提取物中HNF1α与HNF1β蛋白形成复合物结合条带。自身非标记序列竞争探针使目的条带完全消失，以突变的Site1与Site2非标记序列竞争探针，蛋白DNA结合复合物条带不变。为了分析site1与site2与HNF1蛋白结合能力的强弱，以非标记的不同剂量的β-fibrinogen，site1与site2序列竞争β-fibrinogen标记探针，EMSA实验结果显示，上述各序列与HNF1结合能力强弱为：β-fibrinogen>site1>site2。
6．转录因子C/EBP不能调控AFM启动子活性
转录因子C／EBP表达载体与携带有AFM启动子的报告质粒pGL3-mAFM共转染Hep3B细胞系。双荧光素酶报告基因分析显示，peDNA3-C／EBPα或pcDNA3-C／EBPβ单独或联合转染，未改变AFM启动子作用下的荧光素酶强度。
结论：
1．软件分析及缺失报告基因分析，发现AFM基因启动子位于转录起始点上游-336bp～0处。
2．AFM启动子-145处及-75处的HNF1结合位点是AFM启动子的重要组成元件，且site1较site2在AFM启动子转录活性中发挥更重要的作用。
3．EMSA结果提示转录因子HNF1α与HNF1β可直接结合在AFM启动子上两个HNF1结合位点处，双荧光检测系统提示转录因子HNF1α与HNF1β发挥反式激活作用。
4．转录因子HNF1α与HNF1β在维持AFM启动子活性中发挥重要作用，且HNF1β可以抑制HNF1α激活AFM启动子作用。
5．转录因子C／EBP不能激活AFM启动子转录活性。
本研究深入探讨了AFM基因的表达调控机制，为阐明肝特异性基因调节机制奠定了基础，为探索肝脏发育及肝癌发生提供了新的理论依据。
Hepatitis B virus(HBV) is regarded as a major etiological factor for acute or chronic hepatitis,liver cirrhosis and even hepatocellular carcinoma,which are worldwide health problems,especially in China.Clarifying the mechanism of HBV induced HCC will provide a powerful tool to prevent and control the development of HCC.Till now,the pathogenesis for HBV infection is still not clear.However,it is well known integrated HBV DNA might contribute to liver carcinogenesis.Studies found that HBV genome might integrate into the hTERT promoter region in HBV related HCC,in which the hTERT mRNA is transcribed from both the endogenous promoter and integrated HBV gene.
Telomerase is multisubunit complex that forms the ends of eukaryotic chromosomes using its complementary RNA sequence as a template.Telomerase is generally inactive in normal somatic cells but is expressed in most human cancers and immortal cell lines.Recent reports support the concept that the activation of telomerase through transcriptional activation of the hTERT gene is another way by which human tumor-associated viruses can work.
Integrated HBV DNA can encode two types of transcriptional activators,HBx protein and the PreS2 activators.Examination of the viral DNA sequences present in HCC has shown that sequences encoding for the hepatitis B virus X gene(HBx) and/or truncated envelope PreS2/S viral proteins are retained in a large proportion of tumor cells.These two proteins contribute the development of HBV related HCC. However,whether these two proteins may upregulate telomerase activity and the mechanisms are still unknown.Therefore,we firstly developed the study to explore the effects of HBx and PreS2 proteins on telomerase and further study the mechanisms.
Objectives:
1.To examine whether HBx and PreS2 protein regulate tolemerase activity through upregulation of hTERT transcription
2.To map the HBx responsive element in the hTERT promoter
3.To select a minimal region of HBx that is essential for activating hTERT promoter
Methods
1.Effect of HBx and PreS2 protein on telomerase and hTERT gene expression
1.1 Effect of over-expression of HBx and PreS2 protein on telomerase
pcDNA3-HBx or pcDNA3 were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,the protein were extracted from the transfected cells and untransfected cells.Proteins were extracted from HepG2-preS2 cells which were stably transfected with preS2 gene.293 cells were used as a telomerase-positive control.293 cells were heat treated prior as telomerase-negative control.TRAP assay was done to detect telomerase activity.
1.2 Effect of over-expression of HBx and PreS2 protein on hTERT mRNA expression
Semi-quantitative RT-PCR was used to detect hTERT mRNA expression in transfected and untransfected cells.
1.3 Effects of PS-asODNs/HBx,PS-asODNs/preS2 on hTERT mRNA of HepG2.2.15 cells
PS-asODNs/HBx,PS-asODNs/preS2 which specifically targeted to HBx and preS2 were designed and synthesized together with random oligos,PS-rODNs. Different oligos were transfected into HepG2.2.15 cells by lipofectamine.After 48 h, hTERT mRNA was analysed by semi-quantitative PCR.
2.Effect of HBx expression on hTERT promoter
2.1 Constructions of a serial of reporter plasmids containing deleted and mutated hTERT promoter
The specific primers for deleted and mutated hTERT promoter were designed and synthesized for PCR amplification.The PCR products were cloned into pGL3-basic to construct reporter plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.
2.2 Effect of HBx on hTERT promoter
Cultured cells were transfected with pGL3B-TRTP and an increasing amount of pcDNA3-HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
2.3 Mapping of the HBx responsive element in the hTERT promoter
A series of luciferase plasmids containing 5'-truncated hTERT promoter fragments were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBx) into HepG2 cell line.The level of luciferase relative activity was determined 36h after transfection.
2.4 Mapping of the HBx responsive key binding sites in hTERT promoter
A series of luciferase plasmids containing point mutations in c-Myc or Spl binding sites of hTERT promoter were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBX) into HepG2 cell line. The level of luciferase relative activity was determined 36h after transfection.
2.5 Effects of HBx expression on Sp1 gene
Protein and mRNA expression of Sp1,HBx andβ-actin in HepG2-pcDNA3、HepG2-HBx and HepG2 cells were detected by Western blot and semi-quantitative RT-PCR.
3.A minimal region of HBx is essential for activating hTERT promoter
3.1 Constructions of a serial of eukaryotic expression vectors containing truncated HBx gene
The specific primers for truncated HBx were designed and synthesized to amplify truncated HBx genes from pcDNA3-HBx.PCR products were cloned into pcDNA3 to construct serial of truncated HBx expression plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.
3.2 Expression of truncated HBx gene in HepG2 cells
A Serial of truncated HBx vectors were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,proteins were extracted from the transfected cells and untransfected cells.Western blot and RT-PCR were used to detect truncated HBx expression.
3.3 Effect of truncated HBx on hTERT promoter
Cultured HepG2 were transfected with pGL3B-TRTP and a serial of truncated HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control plasmid to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
Results
1.HBx and PreS2 protein upregulate hTERT expression and increase telomerase activity in hepatoma cells
1.1 Over-expression of HBx and PreS2 protein increase telomerase activity
The TRAP assay showed more DNA ladder appeared in HBx and PreS2 transfected cells,which indicated HBx and PreS2 protein increased telomerase activity.
1.2 Over-expression of HBx and PreS2 protein upregulate hTERT mRNA expression
RT-PCR result revealed that hTERT mRNA expression in HBx or PreS2-transfected HepG2 cells was significantly higher than that of pcDNA3 transfected HepG2 cells.There is no significant difference in telomerase activity and hTERT mRNA expression between HepG2 and pcDNA3 transfected HepG2 cells.
1.3 Blockade of HBx and PreS2 downregulate hTERT mRNA expression
RT-PCR confirmed that asODNs/HBx and asODNs/preS2 efficiently suppressed expression of HBx and PreS2 whereas the control rODNs had no affect on HBX and PreS2 expression.As expected,asODNs/HBX and asODNs/preS2 treatment decreased hTERT expression but had not effect onβ-actin expression.
2.HBx regulates Sp1-mediated transcriptional activation of hTERT
2.1 Construction of a serial of deleted and mutated hTERT promoter report vectors
A serial of deleted and mutated hTERT promoter genes were successfully amplified by PCR.The PCR products were cloned into pGL3-basic vector to construct recombinants.The recombinants were transformed into E.coli JM109 and the positive clones were selected by antibiotics,digestion,PCR and sequencing.
2.2 HBx transactivate hTERT promoter significantly
Dual-luciferase report assay indicated that HBx expression vector might transactivate hTERT promoter in HepG2 cells.The luciferase activity of the hTERT promoter was augmented along with the amounts of HBx.The luciferase activity was increased about 1.5-fold greater than the basal promoter activity.The HBx-mediated hTERT promoter activation was detected in other three cell lines SMMC-7721, Be17402 and COS-7 cells.
2.3 The sequences between-197 and+37 in the hTERT promoter show HBx responsiveness.
The luciferase activities of constructs pGL3B-TRTP,pGL3B-895,pGL3B-445, pGL3B-371,pGL3B-306 and pGL3B-197 showed 1.45,1.40,1.46,1.38,1.58 and 1.38-fold induction than basal pormoter,respectively,by HBx.Induction by HBx dropped to the basal level when deletion reached +37(pGL3B+37).All these support that-197 to +37 regions in hTERT promoter are essential for HBx-mediated hTERT activation.
2.4 HBx activates the transcriptional activity of hTERT promoter not through c-Myc,but through Sp1
Effects of HBx on various mutant constructs were initially examined in HepG2 cells.Dual-luciferase report assays showed that HBx up-regulated both wild-type promoter and engineered promoters with mutation in c-Myc binding sites.The results indicate that c-Myc binding sites are not involved in HBx-mediated activation of the hTERT promoter.
We then mutated the five Sp1 sites within the promoter region of the construct pGL3B-del132.Dual luciferase report assay showed that the mutations introduced in Spl sites 2,4 and 5 decreased the induction of the hTERT promoter by HBx.Taken together,these data clearly demonstrate that HBx targets multiple Sp1 binding sites to induce the up-regulation of the hTERT promoter.
2.5 HBx had no effects on the expression of Sp1 in HepG2
Western blot and semi-quantitative RT-PCR showed that there were no significant differences in Spl gene expression between HBx transfected and control HepG2 cells(P＞0.05).
3.The amino acids 50 to 140 of HBx were important for hTERT promoter activation
3.1 Construction of a serial of HBx truncated eukaryotic expression vectors
A serial of truncated HBx genes were successfully amplified by PCR.The PCR products were digested and inserted into pcDNA3-HBx vector.The recombinants were transformed into E.coli DH5αand the positive clones were selected by antibiotics,digestion,PCR and sequencing.
3.2 Truncated HBx expression in HepG2 cells
RT-PCR verified the mRNA expression of all truncated plasmids in HepG2 cells, however,western blot only showed the expression of TC-2,TC-3,HBX,TCN,TN-1 mutants and failed to prove the expression of small mutant,TC-1 and TN-2,probably because of the small size or/and short half-lives.
3.3 The transactivator role of a serial of truncated HBx on hTERT promoter
Dual luciferase report assay showed that hTERT transcriptional activation was diminished in successive deletions of the N-terminal and C-terminal regions of the HBx-protein.In the N-terminal deletion mutant,removal of 50 amino acids did not have any effects on its ability to transactivate hTERT promoter in HepG2 cells. Larger,internal deletion extending from amino acids 1 to 97(TN-2) resulted in disappearance of transactivation.In the analysis of C-terminal deletion mutants, amino acids 140～154 could be deleted from HBx without affecting its ability to transactivate hTERT promoter.However,the HBx transactivating effect was lost in TC-1 and TC-2.Furthermore,removal of 50 amino acids of N-terminal deletion and 141～154 amino acids of C-terminal deletion(TCN) did not change its ability to transactivate hTERT promoter in HepG2 cells.
Conclusion.
1.HBx and PreS2 activator upregulate hTERT expression and increase telomerase activity.
2.HBx stimulates hTERT promoter in a dose-dependent manner in different cells.
3.The hTERT promoter sequences between-197 and+37 show HBx responsiveness in the hTERT promoter.
4.HBx activates the transcriptional activity of hTERT promoter not through c-Myc, but through Sp1.
5.The amino acids 50 to 140 of HBx are important for the transactivation of hTERT promoter.
Our work provides new insights into mechanisms of HBV related HCC and also gives new potential targets for drug design against HBV related HCC.
α-albumin(AFM, also called afamin) is a member of the albumin gene family that includes albumin (Alb), a-fetoprotein (AFP) and vitamin D-binding protein (DBP). Alb family proteins have significant structure similarities and are predicted to have evolved from an ancestral gene. The four genes have all been mapped to the 4q11-q22 region of chromosome and are specifically expressed in the liver. However, the four genes have specific patterns. Alb and AFP are highly induced in hepatoblasts and continue to be expressed at high levels in the fatal liver. Whereas Alb continues to be expressed in the adult liver, AFP is silence during the first several weeks after birth. However the repression is reversible as AFP can be reactivated in the adult liver in situations where there is renewed cell proliferation such as during liver regeneration or in hepatocellular cacinomas. DBP transcription begins mid-gestation in the fatal liver and continues to rise in the adult liver. AFM becomes activated at birth and continues to be highly expressed in the adult liver. Although mechanisms underlying the transcriptional control of the Alb, AFP and DBP have been studied by a number of laboratories, the basic for the tissue-specific and developmentally regulated expression of the AFM gene remains unexplored.
Objectives
1. To analysis the region of AFM promoter and study the promoter activity
2. To find the transcription factor that regulate AFM promoter
Methods
1, Construct a serial of report plasmids containing mouse AFM gene promoter
Full-length mouse AFM promoter sequence and deleted or mutated AFM promoter sequence were respectively cloned into pGL3-basic to construct report plasmids. Cotransfection and Dual-luciferase reporter assay were used to analysis the transcriptional activity of AFM promoter plamids in different cells (Hep3B, HepG2, HEK293 cells) .
2. Regulation of HNF1 on AFM promoter
An increasing amount of HNFl expression vector, pcDNA3-HNFla or pcDNA3-HNFlp was cotransfected into HEK293with pGL3-mAFM respectively cells. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System, in which Renilla luciferase plasmids (pRL-CMV) were cotransfected as a control to standardize the transcription efficiency. All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
3. Regulation of HNFl on mutated AFM promoter
HNFl expression vector (pcDNA3-HNFla or pcDNA3-HNFlp) was cotransfected into HEK293 cells with mutated AFM promoter plasmids. The level ofluciferase relative activity was determined 48h after transfection.
4. Effect of HNFip on HNFla-induced activation of mAFM promoter
Different amount of pcDNA3-HNFip together with O.lug pcDNA3-HNFlawere cotransfected with pGL3-mAFM into HEK293 cells. The level of luciferase relative activity was determined 48h after transfection.
5. Analysis the binding of HNFl protein to HNFl binding sites on AFM promoter
Computer analysis revealed two putative HNF-1 binding sites resided at -145 and -75 of the mAFM promoter, which was named sitel and site2. EMSA and gel supershift assay were involved to examine the actual binding of HNF-1 to the mAFM promoter. Nuclear extracts were prepared from Hep3B cells or HEK293 cells transfected with pcDNA3-HNFla or pcDNA3-HNFip. These extracts were incubated with radiolabeled DNA fragments containing the rat p-fibrinogen promoter HNFl site, sitel fragment, or site2 fragment.
6. Regulation of C/EBP on AFM promoter
C/EBP expression vector pcDNA3-C/EBPa or/and pcDNA3-C/EBPp was/were cotransfected into HEK293 cells with pGL3-mAFM report vector. The level of luciferase relative activity was determined 48h after transfection.
Results
1. A serial of report plasmids containing mouse AFM gene promoter were successfully constructed
Sequencing data of report vectors pGL3-mAFM and deleted or mutated AFM promoter vectors were correspondence with the design. The results of cotransfection and Dual luciferase assays demonstrated that pGL3-mAFM vector showed transcriptional activity in Hep3B and HepG2 cells. In constrast, HEK didn't confer demonstrable transcriptional activity. The findings suggest that AFM transcription is significantly activated in AFM positive cells.
Transcription reduced in promoter activity was observed in constructs extending beyond position -158. However, luciferase activity was almost completely abolished (90% decrease in Hep3B cells) by a further deletion extending to postion -138. Similar results were obtained in mutation of HNF-1 site 1, which suppressed the AFM promoter activity about 90%. The abrogation of the HNF-1 site 2 by substitution of the nucleotide within this region (pGL3-mAFM) reduced transcription by 50% in Hep3B cells. The mutations of both HNF-1 sites completely eliminated the AFM promoter activity, suggesting that HNF-1 might be the most critical factor promoting initiation of AFM transcription.
2. Mouse AFM promoter activity can be up-regulated by HNF-1
The reporter plasmid pGL3-mAFM was co-transfected with increasing doses of pcDNA- HNF1αor 1βplasmid into HEK293 cells which is HNF-1 deficient cells. Normalized luciferase assay results clearly showed that HNF-1 upregulated mouse AFM promoter activity in a dose dependant manner in HEK293 cells, though the action of HNF1βwas only 10% of HNFla.
3. The different activation of HNF1 on mutated mouse AFM promoter activity
To examine the site specificity of mAFM transcriptional activation by HNF-1, these mutated promoter reporter plasmids were cotransfected with HNF1αor 1βexpression plasmids in HEK293 cells. Cotransfection with mock vector was used as control. The mutation at the site lor site2 reduced HNF la-mediated activation to 50% or 40%. The mutation at the sitel didn't reduced HNF IB-mediated activation, while mutation at the site2 reduced HNF IB-mediated activation to 50%. All these suggest that HNF-1-mediated activation of mAFM promoter is primarily but not exclusively dependent on these two HNF-1 sites.
4. The negative effect of HNF1βon HNF1α-induced activation of mAFM promoter
pcDNA3-HNFlp and pcDNA3-HNFla were cotransfected into HEK293 cells with pGL3-mAFM. Dual luciferase assay results clearly showed that HNF1βdecreased HNF1α-mediated mAFM enhancement in a dose-dependent manner.
5. HNF-1αand HNF-1βdirectly binds to the mAFM promoter
EMS A was used to analyze the binding of HNF-1. HNF1 probed bands were detected in the extracts from HEK293 cells transfected with HNF1αor HNF1β, but not mock vector. These bands were also present in Hep3B cell extracts. The bands were completely competed by a homologous competitor, as well as by site 1 or site 2 oligos, but not by the site 1 or site 2 mutated oligos or unrelated oligos. The same results were observed with sitel and site2 probes. When supershift assays were carried, a supershift of specific shifted band was observed with the HNF1αantibody and HNF1βantibody, but not with NF-1 antibody in Hep3B extracts and HEK-293 cells extracts transfected with HNF1α/1βexpression vector.
A modified competition binding study was carried out to extend the observation of the binding of HNF1 toβ-fibrinogen, sitel and site2. Competition for the P~(32)-β-fibrinogen complexes was carried out with 1-, 5-, and 10-fold molar excess of unlabeled p-fibrinogen, sitel and site2 oligos. Signal intensities of the residual bands were quantified. These data revealed that the relative affinities of each HNF1-binding site for HNFl bindings as follows: P-fibrinogen >sitel>site2。
6. C/EBP don't regulate AFM promoter activity
The reporter plasmid pGL3-mAFM was co-transfected with pcDNA-C/EBPa or/and pcDNA-C/EBPp plasmid into Hep3B cells. Dual luciferase assay resultsclearly showed that C/EBP didn't alter mouse AFM promoter activity.
Conclusion:
1. Homologous analysis and reporter assay indicate that AFM promoter resideswithin the 336 bp region from transcriptional initiation site.
2. Two HNF1 sites are found within the AFM promoter region and both HNFl sites appeared to constitute part of the mAFM promoter, but loss of the sitel site had a more profound effect than site2.
3. HNF1αand HNF1βdirectly bind to the mAFM promoter and activate AFM promoter activity.
4. HNF1αand HNF1βare important transcriptional factors for AFM promoter and HNF1βhas negative effect on HNF1α-induced activation of AFM promoter.
5. C/EBP don't regulate AFM promoter activity.
In this paper, we firstly studied the regulation mechanism of a-albumin gene expression. These results may help us to understand how to regulate liver-specific gene expression and provide new theory on mechanism of HCC development.
乙型肝炎病毒(Hepatitis B Virus,HBV)感染肝细胞所致急慢性肝炎及相关肝硬化、肝细胞肝癌(Hepatocellular Carcinoma,HCC)是危害全世界,尤其是我国人民身心健康的重要病因。深入阐明HBV致癌机制,必将为预防和控制肝癌的发生发展提供强有力的手段。目前HBV致癌的确切机理尚未完全清楚,但HBVDNA整合及整合后通过反式激活作用致癌的论点已成为大多数学者的共识。有研究表明在不同的HBV相关HCC组织中,端粒酶编码基因内存在HBV基因片段的整合。这些整合的HBV基因可激活人端粒酶逆转录酶(human telomerasereverse tramscriptase,hTERT),上调端粒酶活性,促进肝癌的发生。
端粒酶(telomerase)位于端粒末端,是一种特殊的核糖核蛋白逆转录酶,大多数正常体细胞不表达端粒酶活性,而在恶性肿瘤中端粒酶活性高表达。研究表明一些病毒编码的蛋白,可通过激活端粒酶来维持端粒长度,阻止细胞死亡,来促进细胞的转化与肿瘤的发生。
HBx与PreS2作为HBV基因编码的两种重要转录激活蛋白,在HBV相关肝癌中的整合率远大于其他蛋白,被认为在HBV相关肝癌的发生发展中起着非常重要的作用。而这两种HBV基因编码的重要转录激活因子,能否上调端粒酶活性,反式激活hTERT启动子促进hTERT转录,以及通过哪些元件激活hTERT启动子,目前还尚未有相关报道。因此,在前期工作的基础上,本课题在国内外率先开展了有关HBV编码反式激活蛋白调控hTERT转录水平的作用及机制研究,为进一步阐明HBV感染相关疾病的发病机制提供新的理论依据。
目的:
1.研究HBx与PreS2蛋白能否促进端粒酶逆转录酶表达,上调端粒酶活性。
2.研究HBx促进端粒酶逆转录酶基因表达的机制,筛选HBx作用于hTERT启动子上关键位点
3.筛选调控hTERT启动子的HBx关键区段
方法:
1.HBx及PreS2蛋白对端粒酶活性及hTERT基因表达的影响
1.1过表达HBx及PreS2蛋白对肝癌细胞端粒酶活性的影响
提取pcDNA3-HBx及空载体对照pcDNA3瞬时转染细胞与未转染对照组HepG2细胞总蛋白;提取稳定表达preS2的HepG2肝癌细胞总蛋白;设立293细胞为端粒酶活性阳性对照组。85℃加热10分钟,灭活293细胞蛋白中端粒酶活性,设为端粒酶活性阴性对照。TRAP法检测端粒酶活性。
1.2过表达HBx及PreS2蛋白对肝癌细胞hTERT基因mRNA水平表达的影响
半定量RT-PCR分别检测pcDNA3-HBx、pcDNA3瞬时转染及未转染HepG2细胞中hTERT mRNA的表达。
半定量RT-PCR分别检测稳定表达preS2的HepG2细胞、pcDNA3空载体对照组细胞及未转染HepG2细胞中hTERT mRNA的表达。
1.3反义封闭HBx与PreS2基因表达对hTERT基因mRNA水平的调控作用
设计合成针对HBx、PreS2的硫代反义寡核苷酸PS-asODNs/HBx、PS-asODNs/preS2及无关对照硫代寡核苷酸PS-asRandom,并以阳离子脂质体介导的方式分别瞬时转染HepG2.2.15细胞系,作用48h后,半定量RT-PCR分别检测各反义寡核苷酸作用组及空细胞对照组hTERT mRNA的表达。
2.HBx对hTERT启动子的调控作用
2.1系列缺失及突变hTERT启动子报告载体的构建
设计、合成针对hTERT启动子不同片段及突变序列的特异性引物,以pCR-TRTP为模板,PCR扩增获得hTERT启动子系列缺失及突变序列的基因。PCR反应产物经酶切定向插入报告基因载体pGL3-basic内,转化大肠杆菌感受态细胞JM109,经抗性筛选、酶切、PCR、DNA测序鉴定获得阳性重组子。
2.2 HBx对全长hTERT启动子的调控作用
将不同剂量的重组子pcDNA3-HBx与携带有hTERT启动子的报告质粒pGL3B-TRTP共转染不同细胞系,同时转染pRL-tk作为内参照。pGL3B-TRTP可在hTERT启动子的作用下表达萤火虫荧光素酶,而pRL-tk可表达海肾荧光素酶。36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析,以均值±标准差表示。
2.3 HBx作用于hTERT启动子关键区域的寻找
将HBx表达载体与系列缺失hTERT启动子报告载体共转染,36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析。
2.4 HBx作用于hTERT启动子关键位点的寻找
将HBx表达载体与系列突变hTERT启动子报告载体共转染,36h后,收集并裂解细胞,测定双荧光素酶表达量,结果进行统计学分析。
2.5 HBx转染前后对Sp1表达水平的影响
提取pcDNA-HBx及pcDNA3瞬时转染与未转染HepG2细胞的各组细胞总蛋白及总RNA,Western blot及RT-PCR检测各组细胞中Sp1,HBx和β-actin的表达水平。
3.调控hTERT启动子的最小HBx关键区域的寻找
3.1截短型HBx重组子的构建
设计、合成针对HBx不同片段基因序列的特异性引物,以pcDNA3-HBx为模板,PCR扩增获得HBx不同截短片段的基因。PCR反应产物经酶切定向克隆入双酶切的pcDNA3-HBx载体内,转化大肠杆菌感受态细胞DH5α,经抗性筛选、酶切、PCR及DNA测序鉴定获得阳性重组子。
3.2截短型HBx在细胞中的表达
系列截短型HBx重组子经脂质体包裹转染HepG2细胞系,48小时后,抽提各组细胞总蛋白与总RNA,Western blot或RT-PCR检测截短型HBx的表达情况。
3.3系列截短型HBx对端粒酶启动子的作用
将系列截短型HBx重组子与携带有hTERT启动子的报告质粒pGL3B-TRTP共转染肝癌细胞系HepG2,同时转染pRL-tk作为内参照。36h后,收集并裂解细胞,测定双荧光素酶表达强度,结果进行统计学分析。
结果
1.HBx及PreS2蛋白上调hTERT mRNA表达,促进端粒酶活性
1.1过表达HBx及PreS2蛋白上调端粒酶活性
TRAP实验显示,在各组细胞中均检测到36bp的内参条带,且条带亮度均一。这说明PCR过程中,Taq酶活性正常,上样量一致。阳性对照组293细胞出现相差6bp的DNA梯形条带,而在阴性对照组,未见梯形条带。稳定或瞬时转染有PreS2或HBx的细胞中DNA梯形条带增多,强度增强,说明HBx及PreS2蛋白过表达可上调端粒酶活性。
1.2过表达HBx及PreS2蛋白促进hTERT基因mRNA表达
半定量RT-PCR结果显示,表达有PreS2或HBx的细胞和对照组细胞相比,hTERT基因mRNA水平明显上调(P＜0.05)。
1.3反义封闭HBx与PreS2基因下调hTERT基因mRNA表达
半定量RT-PCR结果显示,反义寡核苷酸PS-asODNs/HBx、PS-asODNs/preS2能分别有效抑制HBx与PreS2基因的表达。在不影响β-actin基因片段表达的情况下,反义封闭HBx与PreS2基因均可以下调HepG2.2.15细胞中hTERT mRNA的表达。
2.HBx通过Sp1结合位点发挥激活hTERT启动子作用
2.1成功构建系列缺失与突变型hTERT启动子报告载体
PCR成功扩增系列缺失与突变型hTERT启动子基因片段,酶切、连接构建重组报告载体,并成功转化宿主菌,经抗性筛选、酶切鉴定、DNA测序分析获得阳性重组报告载体。
2.2 HBx可反式激活全长hTERT启动子
共转染及双荧光检测系统显示,重组子pCDNA3-HBx作用下的pGL3B-TRTP荧光素酶表达强度较空质粒pcDNA3作用组明显升高,约1.5倍左右(p＜0.05)。HBx对pGL3B-TRTP的激活作用具有剂量依赖性,Western-blot检测显示,随着转染pCDNA3-HBx表达载体剂量的增加,HBx蛋白表达增加,Actin表达没有明显变化,但双荧光检测系统显示荧光素酶表达强度增加。pcDNA3-HBx对pGL3B-TRTP激活作用同样可以在其他三种细胞系Bel7402,SMMC-7721,Cos-7中检测到,说明HBx可以反式激活不同细胞中的hTERT启动子。
2.3 HBx反式激活hTERT的关键区段是启动子-197～+37bp
共转染及双荧光检测系统显示,重组子pCDNA3-HBx作用下的pGL3B-TRTP,pGL3B-895,pGL3B-445,pGL3B-371,pGL3B-306,pGL3B-197报告质粒,其相对荧光素酶表达强度比pcDNA3作用下的表达强度分别增加1.45倍,1.40倍,1.46倍,1.38倍,1.58倍与1.38倍。而hTERT启动子5'端缺失到转录起始点下游37bp时,HBx诱导的荧光素酶表达强度消失。这说明HBx通过hTERT启动子-197～+37区域促进启动子转录。
2.4 HBx通过hTERT启动子上Sp1结合位点发挥激活作用
-197～+37区域内存在5个Sp1结合位点和2个C-myc结合位点。为研究Sp1及C-myc在HBx反式激活hTERT启动子中的作用,构建Sp1结合位点、C-myc结合位点突变的hTERT启动子系列突变报告质粒,并分别与HBx表达载体共转染,结果显示,当单独或联合突变C-myc结合位点时,HBx仍可以促进hTERT启动子作用下的荧光素酶表达强度,说明HBx不是通过C-myc结合位点发挥反式激活作用。当突变了Sp1结合位点2,4,5位时,HBx作用于hTERT启动子的荧光素酶表达强度减弱,与pcDNA3作用组没有明显差异(P＞0.05),说明Sp1结合位点2,4,5位在HBx反式激活hTERT启动子活性中发挥重要作用。
2.5 HBx没有改变Sp1表达水平
Western blot结果,经光密度软件分析,计算Sp1蛋白水平的相对表达强度,结果显示,pcDNA3-HBx转染组与pcDNA3转染组及未转染HepG2细胞组相比,Spl蛋白相对表达强度无显著性差异(P＞0.05)。
半定量RT-PCR结果显示,HBx转染后的HepG2细胞,Sp1 mRNA表达水平与未转染HepG2细胞相比无显著差异(P＞0.05)。
3.激活hTERT启动子的最小HBx基因片段为50-140aa
3.1成功构建系列截短型HBx表达载体
PCR成功扩增系列截短型HBx基因片段,酶切、连接构建重组表达载体,并成功转化宿主菌,经抗性筛选、酶切、PCR、DNA测序分析获得阳性重组子。
3.2系列截短型HBx可在细胞内表达
RT-PCR结果显示,转染有不同截短型HBx肝癌细胞内,可扩增出对应分子量的DNA条带,而在转染pcDNA3的HepG2细胞中没有特异性扩增产物。Western-blot结果显示,转染有不同截短型HBx肝癌细胞内,均见较强的actin蛋白表达,而系列截短型HBx仅检测到TC-2、TC-3、HBX、TCN和TN-1在肝癌细胞HepG2中的表达,未检测到TC-1与TN-2在肝癌细胞中的蛋白表达,这与TC-1、TN-2蛋白分子量较小有关。在转染pcDNA3的HepG2细胞中没有检测到HBx截短型蛋白的表达。
3.3系列截短型HBx对端粒酶启动子的作用
共转染及双荧光检测系统显示,当羧基端缺失14个氨基酸时,即TC-3,HBx仍具有反式激活hTERT启动子作用,当缺失24个氨基酸时,甚或缺失104个氨基酸时,其反式激活作用消失。当氨基端缺失50个氨基酸时,即TN-1,截短HBx仍具有反式激活hTERT启动子作用,但缺失97个氨基酸时,其作用消失。同时在构建的TCN重组载体中也验证了当缺失氨基端前50个氨基酸及羧基端14个氨基酸时,截短型HBx仍具有激活hTERT启动子作用,这说明HBx反式激活hTERT启动子的关键区段在50-140氨基酸区域内。
结论
1.通过过表达及反义封闭实验,证实HBx及PreS2蛋白均可以促进肝癌细胞中hTERT mRNA表达,上调端粒酶活性。
2.HBx在不同细胞系以剂量依赖的方式激活hTERT启动子活性。
3.HBx通过hTERT启动子-197～+37激活其转录表达。
4.HBx通过Sp1结合位点,而不是C-myc结合位点发挥激活hTERT启动子的作用。
5.研究不同的截短型HBx对hTERT启动子的作用,初步找到激活hTERT启动子的HBx关键区段在50～140aa区域内。
意义
该课题的完成不仅为HBV相关HCC机制的阐明提供了新的实验依据,为端粒酶调控研究提供新思路,并为HBV相关HCC的治疗提供新的靶点,具有良好的应用前景。
α白蛋白(a-albumin,又名afamin，简称AFM)是第四个被鉴定的白蛋白家族成员，该家族成员还包含其他三种蛋白：白蛋白(albumin，简称Alb)，甲胎蛋白(a-fetoprotein,AFP)，以及维他命D结合蛋白(Vitamin D-binding protein，DBP)。白蛋白家族成员的编码基因均位于人类第四对染色体的4q11．q22区域或小鼠第十四对染色体上，编码蛋白具有相同的框架结构，在转运血清蛋白中发挥重要的作用，主要在肝组织中表达。但这四个家族成员在肝脏的发育过程有不同的表达形式，因而白蛋白家族是研究肝脏特异性基因表达调控的重要模型。有研究表明AFP及AFM参与肝癌的发生。目前众多国内外学者已经详细研究了如何在转录水平调控Alb、AFP与DBP基因的表达，而关于如何调控AFM基因的表达，研究尚少。
该课题在前期研究的基础上，率先分析了AFM基因启动子活性调控区域，为揭示α白蛋白在肝脏发育过程的调控机制及肝癌发生机制提供新的思路。
目的：
1．分析AFM基因启动子活性区域
2．寻找并研究调控AFM基因转录的调控因子
方法：
1．AFM基因启动子报告质粒的构建与活性分析
分别用双酶切和PCR方法从载体mAFM-LacZ上获得全长与系列缺失或突变的小鼠AFM基因启动子序列，分别克隆到pGL3-basic多克隆位点，构建含有AFM启动子的报告质粒。重组子经酶切、PCR及测序鉴定。将重组子分别转染不同的细胞系Hep3B，HepG2及HEK293细胞，通过检测双荧光素酶表达强度，评估不同的报告质粒中AFM启动子活性。
2．转录因子HNFl对AFM启动子的调控作用
将不同剂量的HNFl真核表达质粒pcDNA3-HNFlQ或pcDNA3-HNFlB单独与AFM启动子报告质粒pGL3-mAFM共转染HEK293细胞，同时转染pRL-CMV作为内参照，检测双荧光素酶表达强度，结果进行统计学分析，以均值±标准差表示。
3．转录因子HNF1对突变型AFM启动子的调控作用
将0．1μg的HNF1真核表达质粒peDNA3-HNF1α或pcDNA3-HNF1β单独与不同突变型AFM启动子报告质粒共转染HEK293细胞，检测双荧光素酶表达强度，结果进行统计学分析。
4．转录因子HNF1α与HNF1β联合对AFM启动子的调控作用
将不同剂量的HNF1β表达载体pcDNA3-HNF1β与0．1μg的HNF1α表达载体pcDNA3-HNFl 0【联合与AFM启动子报告质粒pGL3-mAFM共转染HEK293细胞，检测双荧光素酶表达强度，结果进行统计学分析。
5．EMSA分析转录因子HNFI与AFM启动子相互作用研究
针对TESS软件分析得到的AFM启动子上两个HNFl结合位点site1及site2(-145及-75处)，设计合成分别携带野生型和突变型HNF1反应元件的探针，利用Hep3B与转染有pcDNA3-HNF1α或peDNA3-HNF1β质粒的HEK293细胞核提取物，EMSA分析转录因子HNFl与AFM启动子序列结合情况。
6．转录因子C／EBP对AFM启动子的调控作用
将C／EBP真核表达质粒pcDNA3-CEBPa或／和pcDNA3-CEBPp单独或联合与AFM启动子报告质粒pGL3-mAFM共转染Hep3B细胞，检测双荧光素酶表达强度。
结果：
1．成功构建AFM基因启动子报告质粒
经测序证明成功构建了小鼠系列缺失与突变AFM启动子的报告基因质粒。共转染及双荧光素酶检测系统显示，pGL3-mAFM在Hep3B与HepG2中荧光表达量分别为54．91±3．60与6．70±o．08，而在人胚肾细胞系HEK293中所构建重组子荧光素表达极低2．43±0．17，说明所构建的载体pGL3-mAFM具有特异性的AFM启动子活性。缺失型报告载体pGL3-248与pGL3．158的荧光素酶随着缺失序列的增加，表达强度略有减少，但当缺失了从-158～-138之间的序列，pGL3-138荧光素酶相对表达强度明显下降，下降约90％左右；突变载体pGL3-HNF l mtl荧光素酶表达强度同样下降90％左右。突变载体pGL3-HNF1mt2荧光素酶表达强度下降50％左右，当联合突变HNF1两个结合序列时，pGL3-HNF1mt12荧光素酶强度基本消失，说明转录起始点上游336bp序列是AFM启动子核心序列，HNF-1结合位点是维持AFM启动子活性重要元件。
2．转录因子NNF1对AFM启动子的激活作用
HNF1表达载体pcDNA3-HNF1α或pcDNA3-HNF1β单独与PGL3-mAFM报告载体共转染，随着HNF1α或HNF1β剂量的增加，AFM启动子作用下的荧光素酶表达强度逐渐增加，但HNF1β的激活作用仅为HNF1α作用的10％左右。
3．转录因子HNF1对突变型AFM启动子的激活作用
HNF1表达载体pcDNA3-HNF1α或pcDNA3-HNF1β单独作用于突变型AFM表达载体时，结果显示，当单独突变sitel或site2结合位点时，HNFla作用下的转录活性相比野生型AFM启动子组分别下降50％与40％左右；当单独突变sffel结合位点时，HNF1β作用下的突变型启动子转录活性较野生型AFM转录活性未见改变，而突变site2结合位点时，HNF1β作用下的转录活性较野生型AFM下降50％。说明HNF1α转录因子可通过Site1及site2发挥激活启动子作用，而HNF1β主要通过site2发挥激活AFM启动子作用。当同时突变sitel及site2位点时，HNF1α或HNF1β作用下的转录活性明显下降，与pGL3-basic相比，HNF1α或HNF1β对双突变pGL3-HNF1mt12仍有弱的激活作用，提示在AFM启动子区域存在其他弱的HNF1结合位点。
4．转录因子HNF1β抑制HNF1α激活AFM启动子作用
转录因子HNF1α与HNF1β表达载体与AFM启动子共转染，结果显示随着HNF1β剂量的增加，HNF1α作用下的AFM转录活性逐渐下降。
5．AFM基因启动子区HNF1反应元件与蛋白相互作用分析
EMSA结果显示，AFM启动子上两个HNF1结合位点(site1与site2)都可与核提取物中HNF1α与HNF1β蛋白形成复合物结合条带。自身非标记序列竞争探针使目的条带完全消失，以突变的Site1与Site2非标记序列竞争探针，蛋白DNA结合复合物条带不变。为了分析site1与site2与HNF1蛋白结合能力的强弱，以非标记的不同剂量的β-fibrinogen，site1与site2序列竞争β-fibrinogen标记探针，EMSA实验结果显示，上述各序列与HNF1结合能力强弱为：β-fibrinogen>site1>site2。
6．转录因子C/EBP不能调控AFM启动子活性
转录因子C／EBP表达载体与携带有AFM启动子的报告质粒pGL3-mAFM共转染Hep3B细胞系。双荧光素酶报告基因分析显示，peDNA3-C／EBPα或pcDNA3-C／EBPβ单独或联合转染，未改变AFM启动子作用下的荧光素酶强度。
结论：
1．软件分析及缺失报告基因分析，发现AFM基因启动子位于转录起始点上游-336bp～0处。
2．AFM启动子-145处及-75处的HNF1结合位点是AFM启动子的重要组成元件，且site1较site2在AFM启动子转录活性中发挥更重要的作用。
3．EMSA结果提示转录因子HNF1α与HNF1β可直接结合在AFM启动子上两个HNF1结合位点处，双荧光检测系统提示转录因子HNF1α与HNF1β发挥反式激活作用。
4．转录因子HNF1α与HNF1β在维持AFM启动子活性中发挥重要作用，且HNF1β可以抑制HNF1α激活AFM启动子作用。
5．转录因子C／EBP不能激活AFM启动子转录活性。
本研究深入探讨了AFM基因的表达调控机制，为阐明肝特异性基因调节机制奠定了基础，为探索肝脏发育及肝癌发生提供了新的理论依据。
Hepatitis B virus(HBV) is regarded as a major etiological factor for acute or chronic hepatitis,liver cirrhosis and even hepatocellular carcinoma,which are worldwide health problems,especially in China.Clarifying the mechanism of HBV induced HCC will provide a powerful tool to prevent and control the development of HCC.Till now,the pathogenesis for HBV infection is still not clear.However,it is well known integrated HBV DNA might contribute to liver carcinogenesis.Studies found that HBV genome might integrate into the hTERT promoter region in HBV related HCC,in which the hTERT mRNA is transcribed from both the endogenous promoter and integrated HBV gene.
Telomerase is multisubunit complex that forms the ends of eukaryotic chromosomes using its complementary RNA sequence as a template.Telomerase is generally inactive in normal somatic cells but is expressed in most human cancers and immortal cell lines.Recent reports support the concept that the activation of telomerase through transcriptional activation of the hTERT gene is another way by which human tumor-associated viruses can work.
Integrated HBV DNA can encode two types of transcriptional activators,HBx protein and the PreS2 activators.Examination of the viral DNA sequences present in HCC has shown that sequences encoding for the hepatitis B virus X gene(HBx) and/or truncated envelope PreS2/S viral proteins are retained in a large proportion of tumor cells.These two proteins contribute the development of HBV related HCC. However,whether these two proteins may upregulate telomerase activity and the mechanisms are still unknown.Therefore,we firstly developed the study to explore the effects of HBx and PreS2 proteins on telomerase and further study the mechanisms.
Objectives:
1.To examine whether HBx and PreS2 protein regulate tolemerase activity through upregulation of hTERT transcription
2.To map the HBx responsive element in the hTERT promoter
3.To select a minimal region of HBx that is essential for activating hTERT promoter
Methods
1.Effect of HBx and PreS2 protein on telomerase and hTERT gene expression
1.1 Effect of over-expression of HBx and PreS2 protein on telomerase
pcDNA3-HBx or pcDNA3 were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,the protein were extracted from the transfected cells and untransfected cells.Proteins were extracted from HepG2-preS2 cells which were stably transfected with preS2 gene.293 cells were used as a telomerase-positive control.293 cells were heat treated prior as telomerase-negative control.TRAP assay was done to detect telomerase activity.
1.2 Effect of over-expression of HBx and PreS2 protein on hTERT mRNA expression
Semi-quantitative RT-PCR was used to detect hTERT mRNA expression in transfected and untransfected cells.
1.3 Effects of PS-asODNs/HBx,PS-asODNs/preS2 on hTERT mRNA of HepG2.2.15 cells
PS-asODNs/HBx,PS-asODNs/preS2 which specifically targeted to HBx and preS2 were designed and synthesized together with random oligos,PS-rODNs. Different oligos were transfected into HepG2.2.15 cells by lipofectamine.After 48 h, hTERT mRNA was analysed by semi-quantitative PCR.
2.Effect of HBx expression on hTERT promoter
2.1 Constructions of a serial of reporter plasmids containing deleted and mutated hTERT promoter
The specific primers for deleted and mutated hTERT promoter were designed and synthesized for PCR amplification.The PCR products were cloned into pGL3-basic to construct reporter plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.
2.2 Effect of HBx on hTERT promoter
Cultured cells were transfected with pGL3B-TRTP and an increasing amount of pcDNA3-HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
2.3 Mapping of the HBx responsive element in the hTERT promoter
A series of luciferase plasmids containing 5'-truncated hTERT promoter fragments were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBx) into HepG2 cell line.The level of luciferase relative activity was determined 36h after transfection.
2.4 Mapping of the HBx responsive key binding sites in hTERT promoter
A series of luciferase plasmids containing point mutations in c-Myc or Spl binding sites of hTERT promoter were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBX) into HepG2 cell line. The level of luciferase relative activity was determined 36h after transfection.
2.5 Effects of HBx expression on Sp1 gene
Protein and mRNA expression of Sp1,HBx andβ-actin in HepG2-pcDNA3、HepG2-HBx and HepG2 cells were detected by Western blot and semi-quantitative RT-PCR.
3.A minimal region of HBx is essential for activating hTERT promoter
3.1 Constructions of a serial of eukaryotic expression vectors containing truncated HBx gene
The specific primers for truncated HBx were designed and synthesized to amplify truncated HBx genes from pcDNA3-HBx.PCR products were cloned into pcDNA3 to construct serial of truncated HBx expression plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.
3.2 Expression of truncated HBx gene in HepG2 cells
A Serial of truncated HBx vectors were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,proteins were extracted from the transfected cells and untransfected cells.Western blot and RT-PCR were used to detect truncated HBx expression.
3.3 Effect of truncated HBx on hTERT promoter
Cultured HepG2 were transfected with pGL3B-TRTP and a serial of truncated HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control plasmid to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
Results
1.HBx and PreS2 protein upregulate hTERT expression and increase telomerase activity in hepatoma cells
1.1 Over-expression of HBx and PreS2 protein increase telomerase activity
The TRAP assay showed more DNA ladder appeared in HBx and PreS2 transfected cells,which indicated HBx and PreS2 protein increased telomerase activity.
1.2 Over-expression of HBx and PreS2 protein upregulate hTERT mRNA expression
RT-PCR result revealed that hTERT mRNA expression in HBx or PreS2-transfected HepG2 cells was significantly higher than that of pcDNA3 transfected HepG2 cells.There is no significant difference in telomerase activity and hTERT mRNA expression between HepG2 and pcDNA3 transfected HepG2 cells.
1.3 Blockade of HBx and PreS2 downregulate hTERT mRNA expression
RT-PCR confirmed that asODNs/HBx and asODNs/preS2 efficiently suppressed expression of HBx and PreS2 whereas the control rODNs had no affect on HBX and PreS2 expression.As expected,asODNs/HBX and asODNs/preS2 treatment decreased hTERT expression but had not effect onβ-actin expression.
2.HBx regulates Sp1-mediated transcriptional activation of hTERT
2.1 Construction of a serial of deleted and mutated hTERT promoter report vectors
A serial of deleted and mutated hTERT promoter genes were successfully amplified by PCR.The PCR products were cloned into pGL3-basic vector to construct recombinants.The recombinants were transformed into E.coli JM109 and the positive clones were selected by antibiotics,digestion,PCR and sequencing.
2.2 HBx transactivate hTERT promoter significantly
Dual-luciferase report assay indicated that HBx expression vector might transactivate hTERT promoter in HepG2 cells.The luciferase activity of the hTERT promoter was augmented along with the amounts of HBx.The luciferase activity was increased about 1.5-fold greater than the basal promoter activity.The HBx-mediated hTERT promoter activation was detected in other three cell lines SMMC-7721, Be17402 and COS-7 cells.
2.3 The sequences between-197 and+37 in the hTERT promoter show HBx responsiveness.
The luciferase activities of constructs pGL3B-TRTP,pGL3B-895,pGL3B-445, pGL3B-371,pGL3B-306 and pGL3B-197 showed 1.45,1.40,1.46,1.38,1.58 and 1.38-fold induction than basal pormoter,respectively,by HBx.Induction by HBx dropped to the basal level when deletion reached +37(pGL3B+37).All these support that-197 to +37 regions in hTERT promoter are essential for HBx-mediated hTERT activation.
2.4 HBx activates the transcriptional activity of hTERT promoter not through c-Myc,but through Sp1
Effects of HBx on various mutant constructs were initially examined in HepG2 cells.Dual-luciferase report assays showed that HBx up-regulated both wild-type promoter and engineered promoters with mutation in c-Myc binding sites.The results indicate that c-Myc binding sites are not involved in HBx-mediated activation of the hTERT promoter.
We then mutated the five Sp1 sites within the promoter region of the construct pGL3B-del132.Dual luciferase report assay showed that the mutations introduced in Spl sites 2,4 and 5 decreased the induction of the hTERT promoter by HBx.Taken together,these data clearly demonstrate that HBx targets multiple Sp1 binding sites to induce the up-regulation of the hTERT promoter.
2.5 HBx had no effects on the expression of Sp1 in HepG2
Western blot and semi-quantitative RT-PCR showed that there were no significant differences in Spl gene expression between HBx transfected and control HepG2 cells(P＞0.05).
3.The amino acids 50 to 140 of HBx were important for hTERT promoter activation
3.1 Construction of a serial of HBx truncated eukaryotic expression vectors
A serial of truncated HBx genes were successfully amplified by PCR.The PCR products were digested and inserted into pcDNA3-HBx vector.The recombinants were transformed into E.coli DH5αand the positive clones were selected by antibiotics,digestion,PCR and sequencing.
3.2 Truncated HBx expression in HepG2 cells
RT-PCR verified the mRNA expression of all truncated plasmids in HepG2 cells, however,western blot only showed the expression of TC-2,TC-3,HBX,TCN,TN-1 mutants and failed to prove the expression of small mutant,TC-1 and TN-2,probably because of the small size or/and short half-lives.
3.3 The transactivator role of a serial of truncated HBx on hTERT promoter
Dual luciferase report assay showed that hTERT transcriptional activation was diminished in successive deletions of the N-terminal and C-terminal regions of the HBx-protein.In the N-terminal deletion mutant,removal of 50 amino acids did not have any effects on its ability to transactivate hTERT promoter in HepG2 cells. Larger,internal deletion extending from amino acids 1 to 97(TN-2) resulted in disappearance of transactivation.In the analysis of C-terminal deletion mutants, amino acids 140～154 could be deleted from HBx without affecting its ability to transactivate hTERT promoter.However,the HBx transactivating effect was lost in TC-1 and TC-2.Furthermore,removal of 50 amino acids of N-terminal deletion and 141～154 amino acids of C-terminal deletion(TCN) did not change its ability to transactivate hTERT promoter in HepG2 cells.
Conclusion.
1.HBx and PreS2 activator upregulate hTERT expression and increase telomerase activity.
2.HBx stimulates hTERT promoter in a dose-dependent manner in different cells.
3.The hTERT promoter sequences between-197 and+37 show HBx responsiveness in the hTERT promoter.
4.HBx activates the transcriptional activity of hTERT promoter not through c-Myc, but through Sp1.
5.The amino acids 50 to 140 of HBx are important for the transactivation of hTERT promoter.
Our work provides new insights into mechanisms of HBV related HCC and also gives new potential targets for drug design against HBV related HCC.
α-albumin(AFM, also called afamin) is a member of the albumin gene family that includes albumin (Alb), a-fetoprotein (AFP) and vitamin D-binding protein (DBP). Alb family proteins have significant structure similarities and are predicted to have evolved from an ancestral gene. The four genes have all been mapped to the 4q11-q22 region of chromosome and are specifically expressed in the liver. However, the four genes have specific patterns. Alb and AFP are highly induced in hepatoblasts and continue to be expressed at high levels in the fatal liver. Whereas Alb continues to be expressed in the adult liver, AFP is silence during the first several weeks after birth. However the repression is reversible as AFP can be reactivated in the adult liver in situations where there is renewed cell proliferation such as during liver regeneration or in hepatocellular cacinomas. DBP transcription begins mid-gestation in the fatal liver and continues to rise in the adult liver. AFM becomes activated at birth and continues to be highly expressed in the adult liver. Although mechanisms underlying the transcriptional control of the Alb, AFP and DBP have been studied by a number of laboratories, the basic for the tissue-specific and developmentally regulated expression of the AFM gene remains unexplored.
Objectives
1. To analysis the region of AFM promoter and study the promoter activity
2. To find the transcription factor that regulate AFM promoter
Methods
1, Construct a serial of report plasmids containing mouse AFM gene promoter
Full-length mouse AFM promoter sequence and deleted or mutated AFM promoter sequence were respectively cloned into pGL3-basic to construct report plasmids. Cotransfection and Dual-luciferase reporter assay were used to analysis the transcriptional activity of AFM promoter plamids in different cells (Hep3B, HepG2, HEK293 cells) .
2. Regulation of HNF1 on AFM promoter
An increasing amount of HNFl expression vector, pcDNA3-HNFla or pcDNA3-HNFlp was cotransfected into HEK293with pGL3-mAFM respectively cells. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System, in which Renilla luciferase plasmids (pRL-CMV) were cotransfected as a control to standardize the transcription efficiency. All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.
3. Regulation of HNFl on mutated AFM promoter
HNFl expression vector (pcDNA3-HNFla or pcDNA3-HNFlp) was cotransfected into HEK293 cells with mutated AFM promoter plasmids. The level ofluciferase relative activity was determined 48h after transfection.
4. Effect of HNFip on HNFla-induced activation of mAFM promoter
Different amount of pcDNA3-HNFip together with O.lug pcDNA3-HNFlawere cotransfected with pGL3-mAFM into HEK293 cells. The level of luciferase relative activity was determined 48h after transfection.
5. Analysis the binding of HNFl protein to HNFl binding sites on AFM promoter
Computer analysis revealed two putative HNF-1 binding sites resided at -145 and -75 of the mAFM promoter, which was named sitel and site2. EMSA and gel supershift assay were involved to examine the actual binding of HNF-1 to the mAFM promoter. Nuclear extracts were prepared from Hep3B cells or HEK293 cells transfected with pcDNA3-HNFla or pcDNA3-HNFip. These extracts were incubated with radiolabeled DNA fragments containing the rat p-fibrinogen promoter HNFl site, sitel fragment, or site2 fragment.
6. Regulation of C/EBP on AFM promoter
C/EBP expression vector pcDNA3-C/EBPa or/and pcDNA3-C/EBPp was/were cotransfected into HEK293 cells with pGL3-mAFM report vector. The level of luciferase relative activity was determined 48h after transfection.
Results
1. A serial of report plasmids containing mouse AFM gene promoter were successfully constructed
Sequencing data of report vectors pGL3-mAFM and deleted or mutated AFM promoter vectors were correspondence with the design. The results of cotransfection and Dual luciferase assays demonstrated that pGL3-mAFM vector showed transcriptional activity in Hep3B and HepG2 cells. In constrast, HEK didn't confer demonstrable transcriptional activity. The findings suggest that AFM transcription is significantly activated in AFM positive cells.
Transcription reduced in promoter activity was observed in constructs extending beyond position -158. However, luciferase activity was almost completely abolished (90% decrease in Hep3B cells) by a further deletion extending to postion -138. Similar results were obtained in mutation of HNF-1 site 1, which suppressed the AFM promoter activity about 90%. The abrogation of the HNF-1 site 2 by substitution of the nucleotide within this region (pGL3-mAFM) reduced transcription by 50% in Hep3B cells. The mutations of both HNF-1 sites completely eliminated the AFM promoter activity, suggesting that HNF-1 might be the most critical factor promoting initiation of AFM transcription.
2. Mouse AFM promoter activity can be up-regulated by HNF-1
The reporter plasmid pGL3-mAFM was co-transfected with increasing doses of pcDNA- HNF1αor 1βplasmid into HEK293 cells which is HNF-1 deficient cells. Normalized luciferase assay results clearly showed that HNF-1 upregulated mouse AFM promoter activity in a dose dependant manner in HEK293 cells, though the action of HNF1βwas only 10% of HNFla.
3. The different activation of HNF1 on mutated mouse AFM promoter activity
To examine the site specificity of mAFM transcriptional activation by HNF-1, these mutated promoter reporter plasmids were cotransfected with HNF1αor 1βexpression plasmids in HEK293 cells. Cotransfection with mock vector was used as control. The mutation at the site lor site2 reduced HNF la-mediated activation to 50% or 40%. The mutation at the sitel didn't reduced HNF IB-mediated activation, while mutation at the site2 reduced HNF IB-mediated activation to 50%. All these suggest that HNF-1-mediated activation of mAFM promoter is primarily but not exclusively dependent on these two HNF-1 sites.
4. The negative effect of HNF1βon HNF1α-induced activation of mAFM promoter
pcDNA3-HNFlp and pcDNA3-HNFla were cotransfected into HEK293 cells with pGL3-mAFM. Dual luciferase assay results clearly showed that HNF1βdecreased HNF1α-mediated mAFM enhancement in a dose-dependent manner.
5. HNF-1αand HNF-1βdirectly binds to the mAFM promoter
EMS A was used to analyze the binding of HNF-1. HNF1 probed bands were detected in the extracts from HEK293 cells transfected with HNF1αor HNF1β, but not mock vector. These bands were also present in Hep3B cell extracts. The bands were completely competed by a homologous competitor, as well as by site 1 or site 2 oligos, but not by the site 1 or site 2 mutated oligos or unrelated oligos. The same results were observed with sitel and site2 probes. When supershift assays were carried, a supershift of specific shifted band was observed with the HNF1αantibody and HNF1βantibody, but not with NF-1 antibody in Hep3B extracts and HEK-293 cells extracts transfected with HNF1α/1βexpression vector.
A modified competition binding study was carried out to extend the observation of the binding of HNF1 toβ-fibrinogen, sitel and site2. Competition for the P~(32)-β-fibrinogen complexes was carried out with 1-, 5-, and 10-fold molar excess of unlabeled p-fibrinogen, sitel and site2 oligos. Signal intensities of the residual bands were quantified. These data revealed that the relative affinities of each HNF1-binding site for HNFl bindings as follows: P-fibrinogen >sitel>site2。
6. C/EBP don't regulate AFM promoter activity
The reporter plasmid pGL3-mAFM was co-transfected with pcDNA-C/EBPa or/and pcDNA-C/EBPp plasmid into Hep3B cells. Dual luciferase assay resultsclearly showed that C/EBP didn't alter mouse AFM promoter activity.
Conclusion:
1. Homologous analysis and reporter assay indicate that AFM promoter resideswithin the 336 bp region from transcriptional initiation site.
2. Two HNF1 sites are found within the AFM promoter region and both HNFl sites appeared to constitute part of the mAFM promoter, but loss of the sitel site had a more profound effect than site2.
3. HNF1αand HNF1βdirectly bind to the mAFM promoter and activate AFM promoter activity.
4. HNF1αand HNF1βare important transcriptional factors for AFM promoter and HNF1βhas negative effect on HNF1α-induced activation of AFM promoter.
5. C/EBP don't regulate AFM promoter activity.
In this paper, we firstly studied the regulation mechanism of a-albumin gene expression. These results may help us to understand how to regulate liver-specific gene expression and provide new theory on mechanism of HCC development.
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