Gankyrin介导的去分化在肝癌发生发展中的作用研究
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摘要
研究背景和目的
原发性肝癌是目前世界上最常见的恶性肿瘤之一，其发病率在恶性肿瘤中排名第六，死亡率则居第三。原发性肝癌中肝细胞癌是最常见的类型。肝细胞癌的发生机制较为复杂，主要包括各种致病因素（乙型/丙型肝炎病毒感染、酒精滥用、非酒精性脂肪肝等）引起的肝脏长期慢性炎症与硬化。肝细胞癌的高度异质性导致难以单独采用化疗清除癌细胞，同时手术切除和肝移植也仅限于早期诊断的肝癌。由于大多数肝癌患者诊断时已是中晚期，并且术后频频复发转移，导致肝癌患者的预后较差。肝癌的发病率高、死亡率高、并且缺乏有效的治疗手段，因此研究肝癌发生的机制、探寻新的肝癌预防和治疗靶点就显得尤为重要。
肝细胞去分化在肝癌进展的过程中扮演着重要的作用。肝细胞癌是一个复杂的异质物，其中包含着具有无限增殖潜能的未分化细胞群。近年来，随着对去分化研究的不断深入，通过诱导肝癌细胞分化来治疗肝癌已展现出一定的前景。评价肝癌的分化程度对于肝癌患者的病理诊断、治疗策略的优化选择和预后评估都很关键。
肝细胞结构与代谢功能的维持受到一系列肝富集核因子的交叉调控，这些核因子又称为肝细胞核因子(HNFs，Hepatocyte nuclear factors)，主要包括HNF1α、HNF3β和HNF4α等。在HNF家族中，HNF4α与肝细胞功能和分化状态的维持有重要的联系，也有研究显示，在肝癌细胞中过表达HNF4α，可以在一定程度上诱导肝癌细胞向肝细胞分化。
癌基因p28GANK，又名Gankyrin，也叫做26S蛋白酶体非ATP酶亚基10(PSMD10)，最初发现在肝癌中特异高表达。随着对Gankyrin研究的不断深入，陆续发现它在肺癌、结肠癌等其他类型的恶性肿瘤中也存在高表达。前期研究发现Gankyrin可以与CDK4相互作用促进抑癌蛋白Rb的降解；Gankyrin也可以与MDM2直接结合从而促进MDM2依赖的p53泛素化和降解。近期研究显示，Gankyrin可以通过调控RhoA/ROCK信号通路促进肿瘤发生；另一方面，在DEN诱导的小鼠肝癌模型中，Gankyrin通过促进C/EBPα泛素化降解促进肝癌的发生；另外本实验室的研究也发现Gankyrin可以与RelA结合抑制NF-κB的活性；Gankyrin也能通过活化PI-3K/Akt/HIF1α通路促进Twist1、VEGF、MMP2的表达，从而使肝癌细胞发生EMT，促进肝癌的侵袭转移，影响肝癌病人的预后。而Gankyrin的表达与肝癌去分化之间的关系尚不清楚。因此，本课题研究Gankyrin与肝癌去分化之间的关系及可能的分子机制，有助于解释肝癌的发病机理，并为肝癌的分化治疗提供实验数据。
研究方法：
1.建立大鼠DEN肝癌诱导模型，收集不同病变阶段的大鼠肝脏标本
2.建立小鼠CCl4急性肝损伤模型
3.采用Western-blot、Realtime-PCR、免疫组化等方法研究上述动物模型中Gankyrin及相关分化指标表达的变化
4.在临床肝癌样本中利用免疫组化、Realtime-PCR等方法，研究Gankyrin与去分化指标之间的关系
5.构建Gankyrin相关的干扰及过表达质粒，包装腺病毒和慢病毒，建立稳定细胞系
6.建立大鼠原代肝细胞分离模型，分离原代肝细胞并离体培养，用Gankyrin相关腺病毒进行刺激，研究Gankyrin诱导肝细胞去分化的作用
7.利用CCK8、流式、克隆形成、裸鼠荷瘤等方法，检测Gankyrin相关肝癌稳定细胞系的恶性表型变化
8.采用Realtime-PCR、流式、免疫组织荧光、Spheroid形成实验等方法检测临床样本和肝癌稳定细胞系肝癌干细胞表型的变化
9.利用免疫共沉淀、免疫组化等方法研究Gankyrin对肝细胞核因子的调控作用及机制
研究结果：
1.在CCl4诱导的小鼠急性肝纤维化-硬化模型中发现Gankyrin的表达增加；在大鼠DEN肝癌诱导模型中，Gankyrin的表达随着病变程度的加重而不断升高，并且与去分化相关指标的变化趋势一致
2. Gankyrin在临床样本中的表达与肝癌组织的分化程度负相关
3. Gankyrin可以在体外诱导原代大鼠肝细胞去分化
4.干扰肝癌细胞系中Gankyrin的表达可以抑制其恶性表型，诱导肝癌细胞分化，并减少干扰细胞系裸鼠皮下荷瘤成瘤
5.干扰Gankyrin表达可以抑制肝癌干细胞表型
6. Gankyrin通过诱导HNF4α的降解促进了肝细胞和肝癌细胞的去分化，这种调控作用在体内实验和临床标本中均得到了证实
结论：
本课题从细胞、动物等不同水平证实了Gankyrin在肝癌发生、发展的过程中发挥了重要的作用。我们的研究发现Gankyrin在肝癌发生过程中表达逐渐增强；肝癌中Gankyrin的表达与分化程度负相关；Gankyrin可以通过诱导HNF4α降解引起去分化从而促进肝癌的发生发展；通过慢病毒介导的microRNA干扰Gankyrin，可以有效地诱导肝癌细胞分化、抑制肝癌干细胞表型、使干扰细胞系裸鼠荷瘤成瘤减少。Gankyrin诱导去分化的创新性研究进一步完善了Gankyrin的调控网络，也为临床应用Gankyrin相关抑制剂进行分化治疗和靶向治疗提供了思路和依据。
研究背景和目的
肝癌是最常见的恶性肿瘤之一，其发病率和死亡率长期居高不下。我国是世界上肝癌发病最多的国家，每年新发病例约占全球的50%。目前肝癌的治疗仍以手术治疗为主，但肝癌发病隐匿，相当一部分病人发现时已是中晚期，失去了手术时机；我国还有很多肝癌病人经历了长期的肝硬化病程，全身状况差，不能承受外科治疗；另外，由于尚无有效的化疗药物，大量肝癌病人外科手术后复发，二次手术难度大、风险高。因此，肝癌的预防和治疗一直我国医学研究领域的热点问题。肝癌的发生发展是一个多阶段的复杂过程，已有的研究已经报道了多种癌基因、生长因子等异常表达与肝癌细胞的恶性转化和侵袭转移相关。但目前为止，尚未发现一个理想的预防或治疗肝癌的干预靶点。另外，由于肝癌术后极易复发转移，传统的临床预后指标存在明显的局限性。由此，阐明肝癌发生发展的分子机制，寻找相关标志物和干预靶点是当前肝癌研究的重点和难点。
真核细胞中含有大量的蛋白质，在生物体的生命周期中，各种蛋白质均处于持续动态更新（turnover）的平衡状态，蛋白质的合成与降解处于同等重要的地位。在以往的研究中，很多工作都致力于研究细胞怎样调控特定蛋白质的合成，而对其降解机制研究则较少，然而，越来越多的证据表明，蛋白质的降解途径是一个耗能的，有序的过程，而且受到精细复杂的调控，其中很多关键分子与其他信号通路还存在交互作用（cross-talk）。该系统参与了很多细胞生物学行为的调节，并与许多疾病包括肿瘤的发生发展密切相关。因此研究蛋白降解系统的调控及意义已成为医学领域的一个热点，其中某些关键的调控分子可能成为疾病治疗的靶标，部分靶向蛋白降解系统的药物和治疗方案已经进入应用开发阶段。
细胞内的蛋白质主要通过两种不同的途径降解：溶酶体（lysosome）途径和泛素－蛋白酶系统（The ubiquitin–proteasome system, UPS)）途径，其中，溶酶体主要降解细胞膜上的蛋白，但也可通过自噬途径，降解某些胞浆中的蛋白。相比之下，泛素－蛋白酶体途径则能够高选择性地降解细胞浆中的可溶性蛋白，对细胞正常生理功能的维持具有十分重要的意义。近来研究发现，蛋白泛素化、去泛素化的异常和多种肿瘤的发生密切相关，癌基因和抑癌基因产物的降解异常在很多恶性肿瘤中都存在。一方面，某些癌基因和抑癌基因的泛素化降解失控，会导致癌蛋白的异常富集和抑癌蛋白的减少，最终增加肿瘤发生的风险；另一方面，本实验室前期研究发现，26s蛋白酶体的19s调节复合体的非ATP酶亚基p28GANK(Gankyrin)在肝癌中特异性高表达，并且能够使抑癌基因Rb发生磷酸化而失活，导致转录因子E2F1的活性释放，促进肿瘤进展。这些结果提示，研究UPS中重要分子的功能，不能仅仅局限于蛋白质的降解过程，而更应关注与其他系统或信号通路的交互作用。
我们与美国的合作实验室采用基因表达谱芯片检测70例肝癌患者肿瘤和瘤旁组织样品，利用生物信息学分析，结合Realtime-PCR验证，筛选出在肝癌中异常表达的基因PSMD4（Proteasome26S non-ATPase regulatory subunit4)，该基因编码的蛋白是人26S蛋白酶体非ATP酶亚基4，PSMD4分子含有N端的von Willebrand factorA模序和C端的两段15个氨基酸的泛素相互作用模序（ubiquitin interacting motif，UIM）。
PSMD4在泛素-蛋白酶降解系统中的作用一直备受争议。虽然是最早被鉴定的能够通过UIM与泛素化蛋白质结合的分子，但是接近90%的泛素化蛋白分子的转运和降解并不依赖PSMD4。遗传学研究发现与其他对于酵母生存至关重要的大多数蛋白酶体中其他亚基不同，该基因的缺失并不影响酵母的存活。然而，在拟南芥（Arabidopsis thaliana），黑腹果蝇（Drosophila melanogaster）等高等真核生物中，PSMD4的缺失会导致发育停滞或者死亡，并且PSMD4基因敲除小鼠在胚胎发育早期死亡。研究表明，PSMD4除了作为一个亚基参与蛋白酶体的组装，有很大一部分处于游离状态存在于细胞浆中，这提示该分子有可能具有类似泛素转运体Ubl-UBA（ubiquitin-like, ubiquitin-associated）家族成员Rad23/hHR23, Dsk2/hPLIC/ubiquilin和Ddi1等的功能。虽然普遍认为泛素转运体促进蛋白质降解，但是诸多的证据表明，他们也能够抑制泛素－蛋白酶体系统的活性。体外实验证实Rad23能够剂量依赖地抑制泛素链的合成，体内实验发现在哺乳动物细胞中hPLIC可以抑制p53和IkB的降解。有报道PSMD4与hPLIC-2和hHR23A不仅在遗传学和功能上存在联系，而且分子之间还有相互作用。最近的研究表明，游离的PSMD4能够限制泛素结合蛋白Dsk2与蛋白酶体的结合，维持泛素－蛋白酶体系统的功能，对细胞稳态的平衡具有重要作用。此外，GeethaA等发现，PSMD4能够不依赖泛素地与Inhibitors of DNAbinding（Id1）结合，促进包含basic helix-loop-helix（bHLH）结构域的转录因子活化并与DNA结合，调控细胞分化。关于PSMD4与疾病的关系，已有学者开展了初步的研究，PSMD4肽链的N端能够被水解为40kd左右的片段，该片段被称为Antisecretory Factor1（AF-1），能够强烈抑制肠粘膜的分泌功能，并且与免疫调节和多种肠道病变相关。药物基因组学分析发现，染色体1q21区域的拷贝数扩增与多发性骨髓瘤对bortezomib的耐药和预后不良密切相关，而位于该染色体区域并在此过程中发挥关键作用的基因就是PSMD4。国外学者对肝癌基因表达谱分析提示PSMD4在肝细胞癌中呈现高表达，但并未对该结果做进一步验证和深入分析。以上这些研究结果提示，除了参与细胞蛋白质的降解，PSMD4在高等真核生物的发育和存活等细胞生物学过程中也起到了非常重要的作用，在某些疾病的病程中也有一定的作用，但具体的功能和分子机制尚待阐明。PSMD4在肝癌进展中的作用并不清楚，我们希望通过本课题的研究，阐明PSMD4对肝癌恶性表型的影响及作用机制。
研究方法：
1.收集临床肝癌标本，利用免疫组化等方法，研究PSMD4在肝癌标本中的表达情况
2.利用慢病毒系统建立干扰PSMD4稳定细胞系，采用CCK8、流式、Western-blot、Realtime-PCR等方法研究干扰PSMD4对肝癌恶性表型的影响
3.采用Spheroid形成实验、流式等方法检测PSMD4稳定干扰细胞系肝癌干细胞表型的变化
4.采用常规化疗药顺铂刺激稳定细胞系，研究PSMD4对肝癌细胞化疗敏感性的影响
5.建立大鼠原代肝细胞分离模型，分离原代肝细胞并离体培养，用各种生长因子、炎症因子刺激原代肝细胞，研究这些因子对PSMD4表达的调控作用
6.利用Realtime-PCR、Western-blot等方法研究PSMD4对Ras/Raf/MAPK及PI-3K/Akt信号通路活性的影响
7.利用Realtime-PCR、Western-blot、免疫共沉淀等方法初步研究PSMD4影响化疗敏感性和肝癌干细胞表型的分子机制，并探寻PSMD4可能的作用靶点
研究结果：
1. PSMD4在肝癌临床样本中高表达
2.干扰PSMD4抑制了肝癌细胞的恶性表型和自我更新能力，并减少了肝癌干细胞的比例
4.干扰PSMD4提高了肝癌细胞对常规化疗药顺铂的敏感性
5. PSMD4受到生长因子、炎症因子的调控
6.干扰PSMD4抑制Ras/Raf/MAPK及PI-3K/AKT信号通路的活性
7. PSMD4影响Ras/Raf/MAPK信号通路关键分子Raf-1的表达
8. PSMD4影响了β-catenin、Notch1/3、ABCG2、OCT-4、Sox2等重要干细胞表型相关基因的表达
结论：
本研究通过对大量临床肝癌标本的研究，发现PSMD4在肝癌中高表达。体外研究表明PSMD4能够促进肝癌细胞增殖和克隆形成的能力、增强肝癌干细胞表型、影响肝癌细胞对化疗药的敏感性。初步的分子机制研究证实，PSMD4可能是通过调控Ras/Raf/MAPK通路、Akt通路的活化，并影响β-catenin等重要分子的表达从而影响了肝癌细胞的生物学特性。本课题从临床和细胞水平验证了PSMD4作为肝癌治疗靶点的价值，并初步探讨了PSMD4影响肝癌恶性表型可能的分子机制，最后希望在将来的转基因动物模型中进一步验证其作为肝癌治疗干预靶点的价值。
Hepatocellular carcinoma (HCC) is one of the most common cancers and the thirdmost frequent cause of cancer death in the world. The high heterogeneity of HCC makes itdifficult to eliminate the cancer cells with chemotherapy alone. Meanwhile, surgicalresection and liver transplantation are limited to early stage of HCC. Late diagnosis,recurrence and metastasis result in the poor prognosis of HCC worldwide. Five-yearsurvival rate of patients undergone surgical resection is disappointedly low. It is necessaryto elucidate the underlying mechanisms of hepatocarcinogenesis and identify novel targetsfor HCC prevention and therapy.
Hepatocyte dedifferentiation is a key cellular event during hepatocarcinogenesis,which is featured by alteration of morphology and loss of hepatic function. Hepatoma hasbeen characterized as a heterogeneous complex that includes undifferentiated cells withinfinite proliferative capacity. Induction of hepatoma cell differentiation has beenconsidered as a promising strategy for HCC treatment. Clinicopathological studies havedemonstrated that poorly differentiated HCCs are associated with poorer convalescence,and that a higher differentiation grade of HCCs is related to a lower recurrence rate.Assessment of differentiation grades of HCCs is not only important for the evaluation ofpathological diagnosis and prognosis, but also critical for the optimization of therapeuticstrategies.
The maintenance of liver architecture and hepatic function is cross-regulated by a setof liver enriched nuclear factors termed hepatocyte nuclear factors, which includes HNF1α,HNF3β and HNF4α, etc. Among the members of HNF family, HNF4α is most closelyassociated with the differentiation status of HCC. Our previous study has indicated thatforced re-expression of HNF4α was, to some extent, sufficient to induce the differentiationof hepatoma cells into hepatocytes.
Gankyrin, also named as26S proteasome non-ATPase regulatory subunit10(PSMD10), was reported to be an oncoprotein principally overexpressed in human HCC by our and other studies. It has been documented that the interaction of gankyrin and CDK4facilitates the degradation of Rb. Gankyrin also binds MDM2directly and acceleratesMDM2-dependent ubiquitination and degradation of p53. Fujita at el. showed thatIGFBP-5levels were up-regulated by gankyrin in hepatoma cells and their data suggestedthat gankyrin may play an oncogenic role at the early stages of humanhepatocarcinogenesis. We also reported that gankyrin binds RelA in the nucleus of HCCcells and transports NF-kB back to cytoplasm, thus inhibiting the activation of NF-kB. Ourmost recent data also clarified p28GANKover-expression accelerates hepatocelluarcarcinoma invasiveness and metastasis via PI-3K/Akt/HIF-1α pathways and HCC patientswith high gankyrin expression in general had worse prognosis than those with lowgankyrin expression, which indicates gankyrin could be a candidate gene for riskprognostication of HCC.
However, the role of gankyrin in the differentiation of hepatocytes or hepatoma cellshas not been reported so far. In this study, we clarified, for the first time, that gankyrinexpression was inversely correlated with the differentiation of HCC and over-expression ofgankyrin significantly enhanced the degradation of HNF4α in hepatoma cells, whichindicates that gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells wasat least partially via an HNF4α-dependent mechanism. Moreover, we showed thatdifferentiation induced by gankyrin interference dramatically reduced the proportion ofcancer stem cells (CSCs) in hepatoma cells and induced the differentiation of hepatomacells, implying a novel strategy for HCC prevention and differentiation therapy.
Liver cancer is a malignant tumor with dismal prognosis, and half patients withHCC reside in China. Up to now, surgical resection remains as the primary treatment forpatients with HCC. For those with severe liver cirrhosis, however, alternative therapy isdesirable due to poor physical condition. Recent findings suggested that aberrantexpression of oncogenes and growth factors were closely related with malignanttranformation and metastatic invasion of hepatoma cells, but effective therapeutic targetsof HCC remain obscure. Therefore, it is urgent to illustrate the mechanism underlyinghepatocarcinogenesis and elicit target for prevention therapies as well as prognosticmarkers.
Previous studies preferentially focused on the regulation of particular gene expressionand protein synthesis while little is known about the protein degradation process. It isrecently acknowledged that degradation plays an essential role in protein turnover to helpmaintain cellular balance. Strictly regulated, protein degradation system is involved inmany diseases, including tumor development. Key mediators in the process are believed tobe important targets for treatment and crosstalk with other signaling pathways arecommonly observed.
There are two different degradation pathways in cells: the lysosome pathway and theubiquitin-proteasome pathway. While lysosome is responsible for the degradation ofmembrane proteins and autophagy, the ubiquitin-proteasome system (UPS) is capable ofmaintaining cellular function by selective degradation of cytoplasmic proteins.Malfunction of UPS is frequently observed in tumorigenesis. Dysregulation of proteinubiquitination leads to elevated expression of oncoproteins and down-regulation oftumor suppressors. Notably, the UPS not only functions in protein degradation, but alsocross-talks with other signaling pathways. For instance, our laboratory previouslydemonstrated that p28GANK(also known as Gankyrin, encoded by26S proteasomenon-ATPase regulatory subunit10) was up-regulated in HCC tissues, releasingtranscription factor E2F1via suppressing phosphorylation of Rb.
Our previous studies on mRNA expression microarray of70cases of para-tumortissues and tumor samples of HCC patients demonstrated that PSMD4(Proteasome26S non-ATPase regulatory subunit4) was aberrantly over-expressed in HCC tissues. In ourcurrent study, PSMD4was found to be closely related with malignant transformation viaRas/Raf/MAPKs and PI-3K/Akt signaling cascades in hepatoma cells. This project willfocus on the role of PSMD4in the malignancy of HCC and its molecular mechanismsusing lentivirus-delivered microRNA targeting PSMD4. The results not only shed newlight on the mechanism of hepatocarcinogenesis but also provide new target for diagnosisand treatment of HCC.
原发性肝癌是目前世界上最常见的恶性肿瘤之一，其发病率在恶性肿瘤中排名第六，死亡率则居第三。原发性肝癌中肝细胞癌是最常见的类型。肝细胞癌的发生机制较为复杂，主要包括各种致病因素（乙型/丙型肝炎病毒感染、酒精滥用、非酒精性脂肪肝等）引起的肝脏长期慢性炎症与硬化。肝细胞癌的高度异质性导致难以单独采用化疗清除癌细胞，同时手术切除和肝移植也仅限于早期诊断的肝癌。由于大多数肝癌患者诊断时已是中晚期，并且术后频频复发转移，导致肝癌患者的预后较差。肝癌的发病率高、死亡率高、并且缺乏有效的治疗手段，因此研究肝癌发生的机制、探寻新的肝癌预防和治疗靶点就显得尤为重要。
肝细胞去分化在肝癌进展的过程中扮演着重要的作用。肝细胞癌是一个复杂的异质物，其中包含着具有无限增殖潜能的未分化细胞群。近年来，随着对去分化研究的不断深入，通过诱导肝癌细胞分化来治疗肝癌已展现出一定的前景。评价肝癌的分化程度对于肝癌患者的病理诊断、治疗策略的优化选择和预后评估都很关键。
肝细胞结构与代谢功能的维持受到一系列肝富集核因子的交叉调控，这些核因子又称为肝细胞核因子(HNFs，Hepatocyte nuclear factors)，主要包括HNF1α、HNF3β和HNF4α等。在HNF家族中，HNF4α与肝细胞功能和分化状态的维持有重要的联系，也有研究显示，在肝癌细胞中过表达HNF4α，可以在一定程度上诱导肝癌细胞向肝细胞分化。
癌基因p28GANK，又名Gankyrin，也叫做26S蛋白酶体非ATP酶亚基10(PSMD10)，最初发现在肝癌中特异高表达。随着对Gankyrin研究的不断深入，陆续发现它在肺癌、结肠癌等其他类型的恶性肿瘤中也存在高表达。前期研究发现Gankyrin可以与CDK4相互作用促进抑癌蛋白Rb的降解；Gankyrin也可以与MDM2直接结合从而促进MDM2依赖的p53泛素化和降解。近期研究显示，Gankyrin可以通过调控RhoA/ROCK信号通路促进肿瘤发生；另一方面，在DEN诱导的小鼠肝癌模型中，Gankyrin通过促进C/EBPα泛素化降解促进肝癌的发生；另外本实验室的研究也发现Gankyrin可以与RelA结合抑制NF-κB的活性；Gankyrin也能通过活化PI-3K/Akt/HIF1α通路促进Twist1、VEGF、MMP2的表达，从而使肝癌细胞发生EMT，促进肝癌的侵袭转移，影响肝癌病人的预后。而Gankyrin的表达与肝癌去分化之间的关系尚不清楚。因此，本课题研究Gankyrin与肝癌去分化之间的关系及可能的分子机制，有助于解释肝癌的发病机理，并为肝癌的分化治疗提供实验数据。
研究方法：
1.建立大鼠DEN肝癌诱导模型，收集不同病变阶段的大鼠肝脏标本
2.建立小鼠CCl4急性肝损伤模型
3.采用Western-blot、Realtime-PCR、免疫组化等方法研究上述动物模型中Gankyrin及相关分化指标表达的变化
4.在临床肝癌样本中利用免疫组化、Realtime-PCR等方法，研究Gankyrin与去分化指标之间的关系
5.构建Gankyrin相关的干扰及过表达质粒，包装腺病毒和慢病毒，建立稳定细胞系
6.建立大鼠原代肝细胞分离模型，分离原代肝细胞并离体培养，用Gankyrin相关腺病毒进行刺激，研究Gankyrin诱导肝细胞去分化的作用
7.利用CCK8、流式、克隆形成、裸鼠荷瘤等方法，检测Gankyrin相关肝癌稳定细胞系的恶性表型变化
8.采用Realtime-PCR、流式、免疫组织荧光、Spheroid形成实验等方法检测临床样本和肝癌稳定细胞系肝癌干细胞表型的变化
9.利用免疫共沉淀、免疫组化等方法研究Gankyrin对肝细胞核因子的调控作用及机制
研究结果：
1.在CCl4诱导的小鼠急性肝纤维化-硬化模型中发现Gankyrin的表达增加；在大鼠DEN肝癌诱导模型中，Gankyrin的表达随着病变程度的加重而不断升高，并且与去分化相关指标的变化趋势一致
2. Gankyrin在临床样本中的表达与肝癌组织的分化程度负相关
3. Gankyrin可以在体外诱导原代大鼠肝细胞去分化
4.干扰肝癌细胞系中Gankyrin的表达可以抑制其恶性表型，诱导肝癌细胞分化，并减少干扰细胞系裸鼠皮下荷瘤成瘤
5.干扰Gankyrin表达可以抑制肝癌干细胞表型
6. Gankyrin通过诱导HNF4α的降解促进了肝细胞和肝癌细胞的去分化，这种调控作用在体内实验和临床标本中均得到了证实
结论：
本课题从细胞、动物等不同水平证实了Gankyrin在肝癌发生、发展的过程中发挥了重要的作用。我们的研究发现Gankyrin在肝癌发生过程中表达逐渐增强；肝癌中Gankyrin的表达与分化程度负相关；Gankyrin可以通过诱导HNF4α降解引起去分化从而促进肝癌的发生发展；通过慢病毒介导的microRNA干扰Gankyrin，可以有效地诱导肝癌细胞分化、抑制肝癌干细胞表型、使干扰细胞系裸鼠荷瘤成瘤减少。Gankyrin诱导去分化的创新性研究进一步完善了Gankyrin的调控网络，也为临床应用Gankyrin相关抑制剂进行分化治疗和靶向治疗提供了思路和依据。
研究背景和目的
肝癌是最常见的恶性肿瘤之一，其发病率和死亡率长期居高不下。我国是世界上肝癌发病最多的国家，每年新发病例约占全球的50%。目前肝癌的治疗仍以手术治疗为主，但肝癌发病隐匿，相当一部分病人发现时已是中晚期，失去了手术时机；我国还有很多肝癌病人经历了长期的肝硬化病程，全身状况差，不能承受外科治疗；另外，由于尚无有效的化疗药物，大量肝癌病人外科手术后复发，二次手术难度大、风险高。因此，肝癌的预防和治疗一直我国医学研究领域的热点问题。肝癌的发生发展是一个多阶段的复杂过程，已有的研究已经报道了多种癌基因、生长因子等异常表达与肝癌细胞的恶性转化和侵袭转移相关。但目前为止，尚未发现一个理想的预防或治疗肝癌的干预靶点。另外，由于肝癌术后极易复发转移，传统的临床预后指标存在明显的局限性。由此，阐明肝癌发生发展的分子机制，寻找相关标志物和干预靶点是当前肝癌研究的重点和难点。
真核细胞中含有大量的蛋白质，在生物体的生命周期中，各种蛋白质均处于持续动态更新（turnover）的平衡状态，蛋白质的合成与降解处于同等重要的地位。在以往的研究中，很多工作都致力于研究细胞怎样调控特定蛋白质的合成，而对其降解机制研究则较少，然而，越来越多的证据表明，蛋白质的降解途径是一个耗能的，有序的过程，而且受到精细复杂的调控，其中很多关键分子与其他信号通路还存在交互作用（cross-talk）。该系统参与了很多细胞生物学行为的调节，并与许多疾病包括肿瘤的发生发展密切相关。因此研究蛋白降解系统的调控及意义已成为医学领域的一个热点，其中某些关键的调控分子可能成为疾病治疗的靶标，部分靶向蛋白降解系统的药物和治疗方案已经进入应用开发阶段。
细胞内的蛋白质主要通过两种不同的途径降解：溶酶体（lysosome）途径和泛素－蛋白酶系统（The ubiquitin–proteasome system, UPS)）途径，其中，溶酶体主要降解细胞膜上的蛋白，但也可通过自噬途径，降解某些胞浆中的蛋白。相比之下，泛素－蛋白酶体途径则能够高选择性地降解细胞浆中的可溶性蛋白，对细胞正常生理功能的维持具有十分重要的意义。近来研究发现，蛋白泛素化、去泛素化的异常和多种肿瘤的发生密切相关，癌基因和抑癌基因产物的降解异常在很多恶性肿瘤中都存在。一方面，某些癌基因和抑癌基因的泛素化降解失控，会导致癌蛋白的异常富集和抑癌蛋白的减少，最终增加肿瘤发生的风险；另一方面，本实验室前期研究发现，26s蛋白酶体的19s调节复合体的非ATP酶亚基p28GANK(Gankyrin)在肝癌中特异性高表达，并且能够使抑癌基因Rb发生磷酸化而失活，导致转录因子E2F1的活性释放，促进肿瘤进展。这些结果提示，研究UPS中重要分子的功能，不能仅仅局限于蛋白质的降解过程，而更应关注与其他系统或信号通路的交互作用。
我们与美国的合作实验室采用基因表达谱芯片检测70例肝癌患者肿瘤和瘤旁组织样品，利用生物信息学分析，结合Realtime-PCR验证，筛选出在肝癌中异常表达的基因PSMD4（Proteasome26S non-ATPase regulatory subunit4)，该基因编码的蛋白是人26S蛋白酶体非ATP酶亚基4，PSMD4分子含有N端的von Willebrand factorA模序和C端的两段15个氨基酸的泛素相互作用模序（ubiquitin interacting motif，UIM）。
PSMD4在泛素-蛋白酶降解系统中的作用一直备受争议。虽然是最早被鉴定的能够通过UIM与泛素化蛋白质结合的分子，但是接近90%的泛素化蛋白分子的转运和降解并不依赖PSMD4。遗传学研究发现与其他对于酵母生存至关重要的大多数蛋白酶体中其他亚基不同，该基因的缺失并不影响酵母的存活。然而，在拟南芥（Arabidopsis thaliana），黑腹果蝇（Drosophila melanogaster）等高等真核生物中，PSMD4的缺失会导致发育停滞或者死亡，并且PSMD4基因敲除小鼠在胚胎发育早期死亡。研究表明，PSMD4除了作为一个亚基参与蛋白酶体的组装，有很大一部分处于游离状态存在于细胞浆中，这提示该分子有可能具有类似泛素转运体Ubl-UBA（ubiquitin-like, ubiquitin-associated）家族成员Rad23/hHR23, Dsk2/hPLIC/ubiquilin和Ddi1等的功能。虽然普遍认为泛素转运体促进蛋白质降解，但是诸多的证据表明，他们也能够抑制泛素－蛋白酶体系统的活性。体外实验证实Rad23能够剂量依赖地抑制泛素链的合成，体内实验发现在哺乳动物细胞中hPLIC可以抑制p53和IkB的降解。有报道PSMD4与hPLIC-2和hHR23A不仅在遗传学和功能上存在联系，而且分子之间还有相互作用。最近的研究表明，游离的PSMD4能够限制泛素结合蛋白Dsk2与蛋白酶体的结合，维持泛素－蛋白酶体系统的功能，对细胞稳态的平衡具有重要作用。此外，GeethaA等发现，PSMD4能够不依赖泛素地与Inhibitors of DNAbinding（Id1）结合，促进包含basic helix-loop-helix（bHLH）结构域的转录因子活化并与DNA结合，调控细胞分化。关于PSMD4与疾病的关系，已有学者开展了初步的研究，PSMD4肽链的N端能够被水解为40kd左右的片段，该片段被称为Antisecretory Factor1（AF-1），能够强烈抑制肠粘膜的分泌功能，并且与免疫调节和多种肠道病变相关。药物基因组学分析发现，染色体1q21区域的拷贝数扩增与多发性骨髓瘤对bortezomib的耐药和预后不良密切相关，而位于该染色体区域并在此过程中发挥关键作用的基因就是PSMD4。国外学者对肝癌基因表达谱分析提示PSMD4在肝细胞癌中呈现高表达，但并未对该结果做进一步验证和深入分析。以上这些研究结果提示，除了参与细胞蛋白质的降解，PSMD4在高等真核生物的发育和存活等细胞生物学过程中也起到了非常重要的作用，在某些疾病的病程中也有一定的作用，但具体的功能和分子机制尚待阐明。PSMD4在肝癌进展中的作用并不清楚，我们希望通过本课题的研究，阐明PSMD4对肝癌恶性表型的影响及作用机制。
研究方法：
1.收集临床肝癌标本，利用免疫组化等方法，研究PSMD4在肝癌标本中的表达情况
2.利用慢病毒系统建立干扰PSMD4稳定细胞系，采用CCK8、流式、Western-blot、Realtime-PCR等方法研究干扰PSMD4对肝癌恶性表型的影响
3.采用Spheroid形成实验、流式等方法检测PSMD4稳定干扰细胞系肝癌干细胞表型的变化
4.采用常规化疗药顺铂刺激稳定细胞系，研究PSMD4对肝癌细胞化疗敏感性的影响
5.建立大鼠原代肝细胞分离模型，分离原代肝细胞并离体培养，用各种生长因子、炎症因子刺激原代肝细胞，研究这些因子对PSMD4表达的调控作用
6.利用Realtime-PCR、Western-blot等方法研究PSMD4对Ras/Raf/MAPK及PI-3K/Akt信号通路活性的影响
7.利用Realtime-PCR、Western-blot、免疫共沉淀等方法初步研究PSMD4影响化疗敏感性和肝癌干细胞表型的分子机制，并探寻PSMD4可能的作用靶点
研究结果：
1. PSMD4在肝癌临床样本中高表达
2.干扰PSMD4抑制了肝癌细胞的恶性表型和自我更新能力，并减少了肝癌干细胞的比例
4.干扰PSMD4提高了肝癌细胞对常规化疗药顺铂的敏感性
5. PSMD4受到生长因子、炎症因子的调控
6.干扰PSMD4抑制Ras/Raf/MAPK及PI-3K/AKT信号通路的活性
7. PSMD4影响Ras/Raf/MAPK信号通路关键分子Raf-1的表达
8. PSMD4影响了β-catenin、Notch1/3、ABCG2、OCT-4、Sox2等重要干细胞表型相关基因的表达
结论：
本研究通过对大量临床肝癌标本的研究，发现PSMD4在肝癌中高表达。体外研究表明PSMD4能够促进肝癌细胞增殖和克隆形成的能力、增强肝癌干细胞表型、影响肝癌细胞对化疗药的敏感性。初步的分子机制研究证实，PSMD4可能是通过调控Ras/Raf/MAPK通路、Akt通路的活化，并影响β-catenin等重要分子的表达从而影响了肝癌细胞的生物学特性。本课题从临床和细胞水平验证了PSMD4作为肝癌治疗靶点的价值，并初步探讨了PSMD4影响肝癌恶性表型可能的分子机制，最后希望在将来的转基因动物模型中进一步验证其作为肝癌治疗干预靶点的价值。
Hepatocellular carcinoma (HCC) is one of the most common cancers and the thirdmost frequent cause of cancer death in the world. The high heterogeneity of HCC makes itdifficult to eliminate the cancer cells with chemotherapy alone. Meanwhile, surgicalresection and liver transplantation are limited to early stage of HCC. Late diagnosis,recurrence and metastasis result in the poor prognosis of HCC worldwide. Five-yearsurvival rate of patients undergone surgical resection is disappointedly low. It is necessaryto elucidate the underlying mechanisms of hepatocarcinogenesis and identify novel targetsfor HCC prevention and therapy.
Hepatocyte dedifferentiation is a key cellular event during hepatocarcinogenesis,which is featured by alteration of morphology and loss of hepatic function. Hepatoma hasbeen characterized as a heterogeneous complex that includes undifferentiated cells withinfinite proliferative capacity. Induction of hepatoma cell differentiation has beenconsidered as a promising strategy for HCC treatment. Clinicopathological studies havedemonstrated that poorly differentiated HCCs are associated with poorer convalescence,and that a higher differentiation grade of HCCs is related to a lower recurrence rate.Assessment of differentiation grades of HCCs is not only important for the evaluation ofpathological diagnosis and prognosis, but also critical for the optimization of therapeuticstrategies.
The maintenance of liver architecture and hepatic function is cross-regulated by a setof liver enriched nuclear factors termed hepatocyte nuclear factors, which includes HNF1α,HNF3β and HNF4α, etc. Among the members of HNF family, HNF4α is most closelyassociated with the differentiation status of HCC. Our previous study has indicated thatforced re-expression of HNF4α was, to some extent, sufficient to induce the differentiationof hepatoma cells into hepatocytes.
Gankyrin, also named as26S proteasome non-ATPase regulatory subunit10(PSMD10), was reported to be an oncoprotein principally overexpressed in human HCC by our and other studies. It has been documented that the interaction of gankyrin and CDK4facilitates the degradation of Rb. Gankyrin also binds MDM2directly and acceleratesMDM2-dependent ubiquitination and degradation of p53. Fujita at el. showed thatIGFBP-5levels were up-regulated by gankyrin in hepatoma cells and their data suggestedthat gankyrin may play an oncogenic role at the early stages of humanhepatocarcinogenesis. We also reported that gankyrin binds RelA in the nucleus of HCCcells and transports NF-kB back to cytoplasm, thus inhibiting the activation of NF-kB. Ourmost recent data also clarified p28GANKover-expression accelerates hepatocelluarcarcinoma invasiveness and metastasis via PI-3K/Akt/HIF-1α pathways and HCC patientswith high gankyrin expression in general had worse prognosis than those with lowgankyrin expression, which indicates gankyrin could be a candidate gene for riskprognostication of HCC.
However, the role of gankyrin in the differentiation of hepatocytes or hepatoma cellshas not been reported so far. In this study, we clarified, for the first time, that gankyrinexpression was inversely correlated with the differentiation of HCC and over-expression ofgankyrin significantly enhanced the degradation of HNF4α in hepatoma cells, whichindicates that gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells wasat least partially via an HNF4α-dependent mechanism. Moreover, we showed thatdifferentiation induced by gankyrin interference dramatically reduced the proportion ofcancer stem cells (CSCs) in hepatoma cells and induced the differentiation of hepatomacells, implying a novel strategy for HCC prevention and differentiation therapy.
Liver cancer is a malignant tumor with dismal prognosis, and half patients withHCC reside in China. Up to now, surgical resection remains as the primary treatment forpatients with HCC. For those with severe liver cirrhosis, however, alternative therapy isdesirable due to poor physical condition. Recent findings suggested that aberrantexpression of oncogenes and growth factors were closely related with malignanttranformation and metastatic invasion of hepatoma cells, but effective therapeutic targetsof HCC remain obscure. Therefore, it is urgent to illustrate the mechanism underlyinghepatocarcinogenesis and elicit target for prevention therapies as well as prognosticmarkers.
Previous studies preferentially focused on the regulation of particular gene expressionand protein synthesis while little is known about the protein degradation process. It isrecently acknowledged that degradation plays an essential role in protein turnover to helpmaintain cellular balance. Strictly regulated, protein degradation system is involved inmany diseases, including tumor development. Key mediators in the process are believed tobe important targets for treatment and crosstalk with other signaling pathways arecommonly observed.
There are two different degradation pathways in cells: the lysosome pathway and theubiquitin-proteasome pathway. While lysosome is responsible for the degradation ofmembrane proteins and autophagy, the ubiquitin-proteasome system (UPS) is capable ofmaintaining cellular function by selective degradation of cytoplasmic proteins.Malfunction of UPS is frequently observed in tumorigenesis. Dysregulation of proteinubiquitination leads to elevated expression of oncoproteins and down-regulation oftumor suppressors. Notably, the UPS not only functions in protein degradation, but alsocross-talks with other signaling pathways. For instance, our laboratory previouslydemonstrated that p28GANK(also known as Gankyrin, encoded by26S proteasomenon-ATPase regulatory subunit10) was up-regulated in HCC tissues, releasingtranscription factor E2F1via suppressing phosphorylation of Rb.
Our previous studies on mRNA expression microarray of70cases of para-tumortissues and tumor samples of HCC patients demonstrated that PSMD4(Proteasome26S non-ATPase regulatory subunit4) was aberrantly over-expressed in HCC tissues. In ourcurrent study, PSMD4was found to be closely related with malignant transformation viaRas/Raf/MAPKs and PI-3K/Akt signaling cascades in hepatoma cells. This project willfocus on the role of PSMD4in the malignancy of HCC and its molecular mechanismsusing lentivirus-delivered microRNA targeting PSMD4. The results not only shed newlight on the mechanism of hepatocarcinogenesis but also provide new target for diagnosisand treatment of HCC.
引文
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