【作者】 马晓黎；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 目的通过建立一种全新的、基于基因功能的筛选手段——SMART(Suppression ofMortality by Antisense Rescue Technique)技术，寻找抵抗HDAC抑制剂诱导肿瘤细胞凋亡的效应基因并初步探讨其作用机制。方法250 nmol／LTSA诱导乳腺癌细胞MCF-7凋亡，并收集处于凋亡不同时段的细胞提取mRNA，构建反义cDNA文库。文库DNA随机转入HeLa细胞中，筛选出阳性转染细胞克隆，存活克隆经扩增后提取Hirt DNA，转化感受态细菌并测序。经生物信息学分析后选择感兴趣的基因进行功能验证，并进一步探讨其作用机制。结果成功构建反义cDNA文库，片段插入率大于90％，平均长度约为1．5kb，库容约为2×10~5；运用NCBI blast比对获得76个EST片段；再次重复筛选验证，选定LIV-1基因进行进一步研究。实验显示，HDAC抑制剂可以选择性诱导肿瘤细胞中LIV-1基因的表达，平板克隆形成实验、MTT实验和FACS检测凋亡实验显示下调LIV-1表达可以抵抗HDAC抑制剂诱导的细胞凋亡，这一效应可能与封闭LIV-1后肿瘤细胞内锌离子稳态被破坏有关。结论SMART技术作为一种基于基因功能的筛选手段，可以高通量、特异性的获得在HDAC抑制剂诱发细胞凋亡过程中的效应基因；基因LIV-1可能是HDAC抑制剂抗肿瘤效应的靶基因，下调LIV-1表达可抵抗HDAC抑制剂诱导肿瘤细胞凋亡，这一效应可能与封闭LIV-1后肿瘤细胞内锌离子稳态被破坏有关。背景和目的研究表明，在HDAC抑制剂的作用下，细胞中占总数17％的基因转录水平会发生变化。如此广泛的作用靶点使得HDAC抑制剂在启动某些治疗性反应的同时也启动了某些意想不到的保护性程序，后者严重影响到HDAC抑制剂的最佳临床疗效。目前临床上由于HDAC抑制剂单药用药治疗效能较低，仅被作为辅助用药，与一些传统的一线化疗药联用，达到增强疗效的目的。因此，揭示其低效能背后暗藏的分子机制对推动该类治疗性药物的临床应用具有重要意义。方法和结果通过cDNA芯片分析，我们发现HDAC抑制剂作用后细胞内大量粘附分子表达上调，其中以粘附分子CDl46的上调最为显著。以CD146为代表，通过Realtime PCR、Western blot和免疫组化等方法证实了，在HDAC抑制剂的作用下，多种肿瘤细胞系、原代肿瘤细胞和动物体内CD146被明显上调。将CDl46的单克隆抗体与HDAC抑制剂联用显著增强了HDAC抑制剂的促进凋亡和抑制血管生成的作用，从而抑制肿瘤恶性生长与转移。其作用机制可能与下调PI3K/AKT／mTOR通路的活性有关。结论HDAC抑制剂单药用药治疗效能较低的原因可能是由肿瘤细胞中粘附分子表达增高引起，将粘附分子的单抗与HDAC抑制剂联用可以增强HDAC抑制剂抗肿瘤的生物学效应。这些临床前期研究提示抗粘附剂的使用将对提高HDAC抑制剂效能有很大帮助，为今后的临床治疗提供了一种新的思路和策略。更多还原

【Abstract】 ObjectiveTo construct a noval functional-screening-based method--- SMART （Suppression ofMortality by Antisense Rescue Technique） for the screening of target genes responsible forHDAC inhibitor induced apoptosis and explore the mechanism underlying the action of thegenes.MethodsTo confirm the time course of apoptosis in MCF-7 induced by different concentrationsof Trichostatin A.To induce apoptosis in MCF-7 by treated with 250 nM Trichostatin A.mRNA were extracted from cells collected at multiple time-points after Trichostatin Atreatment.cDNA were synthesized and inserted reversely into plasmid pCEP4 to constrctan anti-sense cDNA library.Library DNA was transfected randomly into Hela,positivetransfected cell clones were screened by Trichostatin A and Hygromycin B.Screening wasstoped when all control cells were died.Surviving cell clones then were amplified andplasmids DNA in cells were acquired by the method of Hirt DNA Extraction.Insertedsequences were then sequenced and EST fragments were obtained.The data were analyzedby the method ofbioinformatics and functional identification.ResultsApoptosis of MCF-7 induced by Trichostatin A started at 36 hours later after drugtreatment and climaxed at about 72 hours.Antisense cDNA library was constructed formcells treated with Trichostatin A within 48 hours at multiple time-points.Insert efficiencywas about 90%,average inserted fragment was about 15 kb and the volum of library wasabout 4×10⁵.Library plasmid DNA were transfected into Hela,after two rounds ofscreening,several cell clones that showed an anti-apoptosis activity were formed.Theseclones were amplified and Hirt DNA was extracted.76 EST fragments were obtained bysequencing.After bioinformatics analysis and functional identification,we acquiredLIV-lgene.Further study showed that these genes knockdown in tumor cell lines couldresist HDACI induced apoptosis,which is caused,at least in part,by disruption ofintracellular zinc homeostasis. ConclusionsSMART cDNA library with high quantity and quality based on HDAC inhibitorinducing apoptosis was successfully constructed;LIV1 gene may be one of the essentialgenes with anti-tumor effects on apoptosis induced by HDAC inhibitors;down-regulationof LIV1 is capable of conferring a significant resistance to HDACi-induced cell death intumor cells,which is caused,at least in part,by disruption of intracellular zinc homeostasis. Evidence for a protective role of CD146 in SAHA-inducederadication of tumorIntroductionHistone deacetylase inhibitors （HDACi） show promise as a novel class of anticanceragents with potent activity to inhibit proliferation,induce apoptosis and differentiation in awide spectrum of tumors.At least 14 HDACi are being tested in over 100 clinical trials anddisplay encouraging therapeutic response with surprisingly good safety profiles^1,2.Theclinical potential of HDACi has been well exemplified by the successful development ofVorinostat （suberoylanilide hydroxamic acid,SAHA）,which has recently been approved bythe US Food and Drug Administration for treating cutaneous T-cell lymphoma³.However,despite the rapid clinical progress achieved,the mechanisms of action of HDACi are not yetwell understood.Early studies using gene profiling techniques have revealed that up to 17%of all known genes are affected by HDACi at the transcriptional level⁴.A large number ofpro- or anti-apoptotic genes and cell cycle regulatory genes have been independentlyidentified by different groups as the downstream targets to HDACi.In addition to HDACs,many non-histone proteins are also regulated by HDACi including transcription factors,nuclear import proteins,signal transduction molecules,cytoskeletal proteins and DNArepair enzymes⁴.One of the central problems with respect to the action of HDACi is thatmodulation of such extensive substrates by these agents might result in the initiation of boththerapeutic responses and unanticipated protective procedures,which significantly limitsthe optimal application of this class of drugs.Identification of the potential protectivesubstrates in HDACi-induced eradication of tumors would set critical biomarkers andfacilitates development of novel HDACi.Recently,a set of clinical data show that there is limited efficacy for HDACi as asingle agent.For example,patients with MDS and AML have had biologic responsesfollowing HDACi therapy;however,true PRs or CRs are infrequent².The majority of thenew trials are combination studies looking at HDACi in combination with other agents^5,6Two PhaseⅠstudies demonstrated that the combination of SAHA with conventionalchemotherapeutic agents such as carboplatin/paclitaxel and FOLFOX regimen （fluorouracil, leucovorin and oxaliplatin） displayed synergistic effect in the treatment of advanced solidmalignancies^7,8.Three PhaseⅠ/Ⅱtrials combining HDACi （PB or VPA） with thedemethylating agents of 5-azacytidine or decitabine in patients with AML and MDSshowed both tolerability and promising efficacy^9-11.Similarly,synergism has also beenobserved between HDACi and other agents such as all-trans-retinoic acid,proteasomeinhibitor,HSP90 antagonists,tyrosine kinase inhibitors and so on^12-15.All of thesecombination trials seek to increase the tumoricidal impact.However,although thesecombination strategies follow a rational molecular approach in some cases;in most cases,they are relatively empirical.Furthermore,unfortunately,synergism in efficacy might beaccompanied by adverse effects that are rarely or never seen with HDACi alone such asmyelosuppression⁶.And high-dose HDACi with substantially longer half-lives show ahigher toxicity that precludes daily treatment¹⁶.Therefore,revealing the molecularmechanisms underlying the low potency of HDACi is pivotal to develop potentially lesstoxic and more effective treatments and promote the optimal application of this class oftherapeutic drugs.In this study we analyzed the HDACi-induced expression pattern using cDNAmicroarrays and confirmed that many of adhesion molecules could be significantlyup-regulated.Interestingly,among these inducible adhesion molecules,CD146 expressionlevel was the highest.To further elucidate whether the increased expression levels ofadhesion molecules influenced HDACi activity we took CD146 for example investigatingthe therapeutic effects of combined treatment with CD146 mAb and HDACi onangiogenesis,tumor growth and metastasis.更多还原