【作者】 刘丽梅；

【导师】 钱程；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2013， 博士

【摘要】 肝癌（Hepatocellular carcinoma，HCC）是最常见的恶性肿瘤之一，全球发病率居恶性肿瘤第五位，死亡率居第三位，肝切除和射频消融对肝癌早期的小肿瘤是目前最好的选择。然而，肝癌患者术后或射频消融后容易复发，并且对放疗、化疗易产生耐药性。因此，肝癌患者预后很差。肿瘤干细胞理论表明，在肿瘤中存在一小群具有干细胞样的肿瘤细胞亚群，具有无限增殖、自我更新、高致瘤性，因此被称为肿瘤干细胞（CSCs）或肿瘤起始细胞（TICs）。目前研究认为，肿瘤干细胞更耐放化疗、具有EMT表型，是术后复发和转移的根源。因此剖析肝癌干细胞自我更新的机制，针对性开发杀伤肝癌干细胞的治疗方法成为当前肝癌研究的重点。本课题组在前期的研究中，已构建以Nanog为标记的肝癌干细胞模型，并指出Nanog的表达是受IGF信号调节。然而，调节肝癌干细胞自我更新的一些内在分子机制仍不清楚。SIRT1兼具调节组蛋白和非组蛋白的乙酰化化水平的活性。既往的研究证实，在实体肿瘤和血液病中，SIRT1的表达增高与病人的进展和预后不良有关。此外SIRT1也能增加对化疗药物的耐药性。而这些现象与肿瘤干细胞的特性高度吻合，提示SIRT1的高表达可能与肿瘤干细胞存在内在联系。基于以上文献回顾和前期研究，我们提出研究假设：去乙酰化酶SIRT1可能参与了肝癌干细胞自我更新的维持。本课题致力研究和探讨SIRT1参与肝癌干细胞自我更新维持的分子机制。本课题主要分三部分进行：第一部分检测SIRT1在肝癌干细胞的表达，并进一步采用体内和体外实验模型，研究SIRT1在肝癌干细胞自我更新中所发挥的作用。第二部分探讨SIRT1维持肝癌干细胞自我更新的分子机制，主要研究SIRT1对关键干细胞转录因子的调节及其分子机制。第三部分是在前两部分的基础上，进一步探讨SIRT1在临床标本的表达以及联合肝癌的小分子化合物多吉美（索拉非尼）对肝癌病人预后和治疗的意义。研究内容和主要结论如下：一、在NanogPos的肝癌干细胞中， SIRT1的表达显著高于NanogNeg的非肝癌干细胞（1）在肝癌干细胞中SIRT1高表达，在非肝癌干细胞SIRT1低表达；（2）在肝癌干细胞分化过程中，SIRT1表达水平呈显著下降趋势；（3）在肝癌干细胞的NOD/SCID小鼠移植瘤中，SIRT1的表达显著高于非肝癌干细胞的NOD/SCID小鼠移植瘤。二、在NanogPos肝癌干细胞中，干扰SIRT1的表达能够抑制肝癌干细胞的自我更新能力并延长NOD/SCID小鼠的生存时间（1）在肝癌干细胞中，干扰SIRT1后能显著抑制肝癌干细胞的增殖；（2）在肝癌干细胞中，干扰SIRT1的表达可显著地降低肝癌干细胞的自我更新能力；（3）在肝癌干细胞中，干扰SIRT1的表达可明显抑制NOD/SCID小鼠移植瘤的成瘤体积和重量；（4）在肝癌干细胞中，干扰SIRT1的表达能够延长NOD/SCID小鼠的存活时间。三、在NanogNeg非肝癌干细胞中，过表达SIRT1能够恢复肝癌干细胞的自我更新能力（1）采用腺病毒过表达SIRT1，可显著地提高非肝癌干细胞的克隆形成和成球形成能力；（2）采用腺病毒过表达SIRT1，可明显增加非肝癌干细胞在NOD/SCID小鼠体内的成瘤率。四、在NanogPos肝癌干细胞中，SIRT1通过SOX2调控肝癌干细胞自我更新（一）在NanogPos肝癌干细胞中，干扰或抑制SIRT1的表达或活性可抑制SOX2的表达（1）在肝癌干细胞中干扰SIRT1的表达或抑制SIRT1的活性能显著下调SOX2的表达，并且是早期发生事件；（2）在非肝癌干细胞中过表达SIRT1，能显著上调SOX2的表达；（3）在临床肝癌组织样本和肝癌细胞系中，SIRT1和SOX2的表达水平呈正相关；（二）在NanogPos肝癌干细胞干扰SIRT1同时过表达SOX2，能够恢复肝癌干细胞的自我更新能力和成瘤能力（1）在肝癌干细胞干扰SIRT1同时过表达SOX2，SOX2的蛋白表达水平显著升高；（2）在肝癌干细胞干扰SIRT1同时过表达SOX2，肝癌干细胞的克隆形成和成球能力显著增加；（3）在肝癌干细胞干扰SIRT1同时过表达SOX2能恢复在NOD/SCID小鼠移植瘤成瘤能力；（三）在NanogPos的肝癌干细胞中，SOX2是SIRT1调控肝癌干细胞自我更新的下游效应分子（1）在肝癌干细胞中，同时干扰SIRT1和SOX2与单独干扰SIRT1或SOX2相比，并不能显著降低SOX2的蛋白表达水平；（2）在肝癌干细胞中，同时干扰SIRT1和SOX2与单独干扰SIRT1或SOX2相比，其克隆形成和成球形成能力无显著性差异。五、在NanogPos肝癌干细胞中，SIRT1通过DNMT3A和组蛋白修饰调节SOX2的表达1.在NanogPos肝癌干细胞中，干扰SIRT1的表达，影响SOX2启动子的转录活性（1）干扰SIRT1的表达能降低SOX2启动子的转录活性；（2）干扰SIRT1后，能够增加5-甲基胞嘧啶在SOX2启动子的修饰水平，但同时下调5-羟甲基胞嘧啶的修饰水平；（3）干扰SIRT1后，SOX2启动子区DNA甲基化水平显著升高；（4）在肝癌干细胞中，干扰SIRT1后加DNA甲基化抑制剂5-氮胞苷(5-Aza’-2)能够恢复SOX2的表达。2.在NanogPos肝癌干细胞中干扰SIRT1， SOX2启动子区H3K4Me3水平下调，而H3K27Me3水平升高；3.在NanogPos肝癌干细胞中， DNMT3A是SIRT1调节SOX2表达的重要表观遗传调控因子（1）干扰SIRT1的表达，相比与DNMT1和DNMT3B，DNMT3A显著增加；（2）干扰SIRT1的表达，DNMT3A的表达随着时间逐渐增加；（3）ChIP实验表明在干扰SIRT1的情况下，DNMT1和DNMT3A结合到SOX2启动子的能力显著增强。4.在NanogPos肝癌干细胞中，SIRT1通过调控DNMT3A的乙酰化修饰影响其表达活性（1）在肝癌干细胞中，SIRT1和DNMT3A相互结合，存在于同一个复合物上；（2）在肝癌干细胞中，干扰SIRT1后，DNMT3A的乙酰化水平显著升高。六、在NanogPos肝癌干细胞中，SIRT1通过SOX2影响Nanog的表达（1）在肝癌干细胞中干扰SIRT1可降低SOX2结合到Nanog启动子的活性；（2）在肝癌干细胞中干扰SIRT1或加SIRT1的抑制剂-TV-6，可降低GFP或Nanog的表达；（3）皮下移植瘤中，干扰SIRT1同时过表达SOX2可恢复GFP或Nanog的表达；（4）SOX2结合到Nanog的启动子进而激活转录。七、SIRT1与SOX2在肝癌肿瘤组织中高表达，两者联合能够作为肝癌患者预后的一个独立指标（1） SIRT1表达的阳性信号主要定位在癌细胞的细胞核，SIRT1在癌组织表达阳性率为62%（92/148），而在相对应的癌旁中的阳性率为31%（48/148）；SIRT1与肝癌的组织学分级、包膜浸润、血管癌栓具有显著差异（p=0.003、p=0.015、p=0.005），并且与患者的预后相关；（2）SOX2表达的阳性信号主要定位在癌细胞的细胞核，SOX2在肝癌在癌组织中表达阳性率为32.9%（49/149）,而在相对应的癌旁中的阳性率为6.7%（10/148）；SOX2与肝癌的血管癌栓、癌内间质增生、肝内转移具有显著差异（p=0.000、p=0.048、p=0.003），并且与患者的预后相关；（3）SIRT1与SOX2在肝癌的表达比值可能是预测肝癌患者预后的一个重要指标，SIRT1和SOX2的表达双阳性组，肝癌病人的预后更差；SIRT1和SOX2的表达双阴性组，肝癌病人的预后好，生存时间长；SIRT1和SOX2的表达与患者的复发、组织学级别、肝内转移有显著性差异（p=0.032、p=0.028、p=0.0084）。八、在NanogPos肝癌干细胞中，SIRT1对化疗药物-索拉菲尼耐药性的研究（1）在肝癌干细胞中，通过干扰或用抑制剂处理降低SIRT1表达或活性后，同时用索拉非尼处理其肝癌干细胞克隆耐受能力显著低于对照组；（2）先用2.5uM TV-6处理细胞6d，再用2.5uM索拉菲尼处理的细胞9d，其肝癌干细胞克隆耐受能力显著低于对照组;（3）干扰SIRT1联合索拉菲尼处理，能更有效地抑制NOD/SCID小鼠移植瘤肿瘤的生长；（4）干扰SIRT1联合索拉菲尼，与对照组相比，其细胞损伤更为严重：NOD/SCID小鼠移植瘤的病理形态学和免疫组化进一步证实抑制SIRT1联合索拉非尼对NOD/SCID小鼠移植瘤肿瘤的坏死率更广泛。九、SIRT1的表达水平可预示临床肝癌患者术后对索拉非尼治疗的敏感性在肝癌样本中SIRT1低表达或不表达患者接受索拉菲尼治疗，其生存时间显著高于SIRT1高表达的对照组。综上所述，本研究的主要结论是：1.去乙酰化酶SIRT1在维持肝癌干细胞中自我更新和高致瘤的特性中具有重要的作用。2.去乙酰化酶SIRT1通过影响DNMT3A的乙酰化进而改变SOX2的启动子活性，从转录水平调节SOX2的表达，参与维持肝癌干细胞自我更新和高致瘤性的特性。3. SIRT1上调SOX2后，可进一步激活Nanog，组成“干性”调控网络，维持肝癌干细胞自我更新和高致瘤的特性。4. SIRT1可作为治疗肝癌的潜在分子靶标。本研究首次报道了去乙酰化酶SIRT1在肝癌干细胞发生过程中的表达变化、以及维持肝癌干细胞自我更新的作用机制，从肝癌治疗的角度探讨了肝癌干细胞耐药的分子机制。研究发现其维持肝癌干细胞维持自我更新的分子机制为：SIRT1通过调控DNMT3A影响SOX2启动子区DNA甲基化进而激活其转录而实现的。在此基础上，提出SIRT1可作为治疗肝癌的一个潜在分子靶标。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the fifth most prevalent tumor and the thirdlethalcause of cancer-related deaths worldwide. Although surgery and radiofrequency ablation(RFA) are the best option for small tumors at early stage of HCC patients, the tumorrecurrence and resistance to chemotherapy and radiotherapy result in poor outcome of HCC.Studies have demonstrated that a subpopulation of cancer cells, often referred to ascancer stem cells (CSCs) or tumor initiating cells (TICs), are capable of extensiveproliferation, self-renewal, and the increased frequency of tumor formation. The concept ofCSCs has significant clinical implications: CSCs have been shown to be more resistant tochemotherapy and radiotherapy; CSCs have the properties of EMT, metastasis and tumorrelapse after surgery. Therefore, understanding common mechanisms in self-renewal of CSCswill enable further research to focus efforts on therapeutic targets which may be most usefulin developing new approaches of treatment. Our previous study have identified transcriptionfactor Nanog as a novel marker for liver CSCs and demonstrated that Nanog could play acrucial role in regulating self-renewal of liver CSCs via IGF signaling pathway. Nevertheless,the key components and molecular mechanisms contributing to biology of liver CSCs arelargely unknown.In cancer, high expression of SIRT1(Sirtuin1, silent mating type information regulation2homolog-1) has been linked with aggressive progression and poor prognosis of leukemiaand solid tumors. Moreover, SIRT1not only regulates the Histone deacetylase andnon-histone proteins, but promotes chemotherapy resistance by enhancing efflux of drugs, aswell as renders cancer cells resist to the radiation-induced apoptosis. The similarities betweenCSCs and SIRT1enable us to put forward a hypothesis that SIRT1may participate theregulation of liver CSCs. In this study, we will explore the expression and biological functionof SIRT1in liver CSCs.In order to identify the relation between CSCs and SIRT1, we divided this study into three sections. The first section was to examine the SIRT1expression pattern in liver CSCs,then to clarify its functional role for maintenance of self-renewal in vitro and in vivo. Thesecond section was to verify that SIRT1regulated transcription of SOX2in liver CSCs. Thethird one was to discuss the SIRT1expression with drug resistance and survival in liver CSCsand HCC patients.A list of expression date and main conclusions are shown as following1. SIRT1is highly expressed in liver CSCs compared to non-CSCs, suggesting thatthe expression of SIRT1is essential for maintaining growth and progression of liverCSCs.(1) Expression of SIRT1in NanogPoscells of liver CSCs was significantly higher thanthose in NanogNegcells of non-CSCs.(2) Expression of SIRT1in NanogPoscells derived from xenograft tumors was higherthan that in NanogNegcells derived from xenograft tumors in NOD/SCID mice.(3) SIRT1is decreasing during the differentiation of liver CSCs in vitro.2. Knock-down of SIRT1expression in the NanogPoscells inhitiors self-renewal ofliver CSCs and prolong survival times in NOD/SCID mice.(1) Knock-down of SIRT1expression in the NanogPoscells significantly inhibit cellgrowth of liver CSCs.(2) Knock-down of SIRT1expression in the NanogPoscells was significantly inhibitcolony and sphere formation efficiency of liver CSCs, and the difference was statisticallysignificant.(3) Knock-down of SIRT1expression could significantly inhibited tumor’s growth, sizeand weight when subcutaneously implanted in NOD/SCID mice.(4) Kaplan-Merier analysis shown that knock-down SIRT1expression could prolongsurvival times in NOD/SCID mouse compared with control group．3. Overexpression of SIRT1restore self-renewal characteristics in liver non-CSCs.(1) Overexpression of SIRT1in the NanogNegcells increased the efficiencies of colonyand sphere formation significantly.(2) Survival rate of tumors was significantly increased in NOD/SCID mice implantedwith NanogNegcells infected with adenoviral vector expressing SIRT1.4. SIRT1control SOX2expression to derive self-renewal characteristics in liver CSCs.(1) Knock-down of SIRT1in the NanogPoscells was decreased SOX2expression in liverCSCs.1) Knock-down of SIRT1in the NanogPoscells was significantly inhibit SOX2expression levels of liver CSCs; Overexpression of SIRT1in the NanogNegwas significantlyincreased SOX2expression.2) Knock-down SIRT1in the NanogPoscells was decreased SOX2expression and itoccurred in the earlier time than Nanog of liver CSCs.3) Western blot results showed that SIRT1and SOX2were positive correlation in HCCcell lines and patients tumor tissues..(2) Overexpression SOX2in knock-down SIRT1cells could be restore theself-renewal characteristics in liver CSCs.1) Immunofluorescence and western blot datas showed that overexpression SOX2in theNanogPoscells with silencing SIRT1was increased SOX2expression level.2) Overexpression SOX2in NanogPoswith silencing of SIRT1that colony and sphereefficiency were significantly increased compared to silencing SIRT1in liver CSCs.3) Overexpression of SOX2in the NanogPoscells with silencing of SIRT1could generatetumor more efficiently as compared to silencing SIRT1in liver CSCs.(3) Co-knockdown SIRT1and SOX2expression did not significantly decreased SOX2expression level, and colony and sphere formation efficiency in liver CSCs.5. SIRT1regulates SOX2expression via DNMT3A（1）SOX2changes by knockdown SIRT1contributes to the transcription regulation inliver CSCs.1）PSOX2-luc luciferase system assay shown that silencing of SIRT1in NanogPoscellsexpressed decreased levels of SOX2.2) ChIP analysis shown that that NangPoscells displayed a relative enrichment oftrimethylated H3K4(H3K4Me3) compared to the levels with silencing of SIRT1. Meanwhile,the levels of trimethylation of H3K27(H3K27Me3) was an increased in silencing of SIRT1compared to CSCs.3) MeDIP assay shown that the increased level of5mC and decreased of5hmC in liverCSCs with silencing of SIRT1. 4) BSP-PCR assay that the CpGs was a significantly increased in non-CSC or withsilencing of SIRT1relative in liver CSCs.5) Western-blot assay showen that SOX2expression was restored in response to5-azacytidine compared with silencing of SIRT1in liver CSCs.(2) DNA methylation is one of the important epigenetic modifications, knockdownSIRT1was increased SOX2DNA methylation in liver CSCs.1) qRT-PCR and western blot assay showed that with silencing of SIRT1expression inliver CSC resulted in significant increased DNMT3A expression.2) Western blot analysis demonstrated that increase expression of DNMT3A in atime-dependent manner with silencing of SIRT1.3) ChIP analysis showed that DNMT3A and DNMT1bound to SOX2promoter moreefficiently in liver CSC with silencing of SIRT1.(3) SIRT1effectively inhibits DNMT3A acetylation and expression in liver CSCs.1) Co-IP analysis shown that SIRT1interacts with DNMT3A in liver CSCs.2) DNMT3A acetylation relative expression was increased with silencing of SIRT1compared with liver CSCs.6. Modulation of Nanog expression by SIRT1is mediated by SOX2(1) PNanog-luc luciferase system assay shown that silencing of SIRT1in NanogPoscellsresulted in the reduced expression of GFP in NanogPoscells with silencing of SIRT1in liverCSCs.(2) Western blot assay shown that there was decreased expression of GFP in NanogPoscells with silencing of SIRT1in liver CSCs.(3) Western blot assay shown that there was decreased expression of GFP in xenografttumors derived from NanogPoscells with silencing of SIRT1.(4) ChIP analysis shown that SOX2can bind to Nanog promoter, with silencing ofSIRT1reduced SOX2binding to Nanog promoter capacity and overexpression of SOX2increased this ability.7. High SIRT1and SOX2expression correlates with poor prognosis in human HCCpatients.(1) Immunohistostaining analysis shown that SIRT1protein was mainly localized in thenucleus of cancer cells, positive rates of SIRT1expression in HCC cancerous tissue was62% (92/148). In contrast, SIRT1expression in adjacent noncancerous tissue was31%(48/148).Clinical and pathological analysis of HCC samples, it is showed that expression of SIRT1inHCC was correlated with tumor stage, capsular invasion and vascular thrombus.Kaplan-Merier analysis demonstrated that SIRT1expression in HCC was significantlycorrelated with overall and disease-free survival.(2) Immunohistostaining analysis shown that SOX2protein was mainly localized in thenucleus of cancer cell, and positive of SOX2expression in HCC was33.1%(49/148). Incontrast, SOX2expression in adjacent noncancerous tissue was6.76%(10/148). Clinical andpathological analysis of HCC samples, and results shown that SOX2expression in HCC wascorrelated with tumor interstitial hyperplasia of tumor、intrahepatic metastasis and vascularthrombus. Kaplan-Merier analysis demonstrated that SOX2expression in HCC wassignificantly correlated with overall and disease-free survival.(3) Kaplan-Merier analysis shown that SIRT1and SOX2expression in HCC wassignificantly correlated with overall and disease-free survival. Moreover, when patients werecombination of high SIRT1and SOX2expression has the poorest outcome compared with theother four possible combinations.8. Fundamental function of SIRT1on drug resistance of liver CSCs(1) The survival of liver CSCs was significantly reduced drug resistant clones when TV6or silencing of SIRT1expression was combined with sorafenib, as compared with that inNanogPoscells. Immunofluorescence analysis shown that treatment with sorafenib couldinduce higher level of DNA damage marker γ-H2AX focis with siliencing ofSIRT1expression compare with NanogPoscell.(2) Liver CSCs were treated with TV6or sorafenib could induce drug resistant clones. Incontrast, treatment of liver CSCs by combination of either of TV-6with sorafenib couldcompletely abolish drug resistant clones.(3) The treatment with sorafenib resulted in inhibition of tumor growth in tumor-bearinganimals with NanogPoscells. In contrast, treatment with sorafenib could eradicate tumors intumor-bearing animals with NanogPoscells with silencing of SIRT1expression. Pathologicaland immunohistochemistical analysis shown that dramatic necrosis and high levels ofγ-H2AX were observed in tumors derived from NanogPoscells with silencing of SIRT1expression. 9. Association SIRT1expression with sorafenib treatment with poor prognosis ofHCC patients.Among31HCC cases, Kaplan-Merier analysis shown that positive SIRT1expressionwith sorafenib resulted was significantly correlated with overall and disease-free survival，patients had poor survival time. In contrast, negative SIRT1expression with sorafenibresulted was significantly correlated with overall and disease-free survival, patients hadlonger survival time.In conclusion, the above data suggest that1. SIRT1as a key regulator in the maintenance of self-renewal and tumorigenic potentialin liver CSCs.2. SIRT1maintains self-renewal and tumorigenic potential of liver CSCs through directregulation of SOX2expression.3. The up-regulation of SOX2by SIRT1and subsequently activate the network of Nanogmay regulate self-renewal and tumorigenic potential of liver CSCs.4. SIRT1serves as a potent molecular target for therapy in liver cancer.In summary, our study reported for the first time that the role and the maintenance ofself-renewal mechanisms of SIRT1in liver CSCs, moreover to discuss the function of SIRT1on drug resistance of liver CSCs. We suggested that high levels of SIRT1are needed tomaintain the undifferentiated state of human liver CSCs, which is achieved through tightcontrol of the levels and binding ability of DNM3A and histone modifiers at the regulatoryregions of pluripotency transcription factor SOX2gene. SIRT1may serve as attractivetherapeutic target for treatment of HCC by targeting liver CSCs.更多还原