【作者】 叶轲；

【导师】 王志明；

【作者基本信息】 中南大学 ， 普通外科学， 2010， 博士

【摘要】 目的：肝细胞癌(HCC)在中国和亚非其他国家是一种常见的恶性肿瘤,在中国其死亡率居各种恶性肿瘤死亡率的第二位。迄今为止,大量研究显示HCC中存在很多畸变表达基因,然而,能够帮助识别早期肿瘤复发并可作为潜在治疗靶点的分子标志物仍然很有限。神经上皮细胞转化基因1(neuroepithelial cell transforming gene 1, Net1)最初是从神经上皮瘤中分离鉴定出来的。其突变型可以转化NIH3T3细胞,因此被认为是癌基因。Net1蛋白是RhoA蛋白特异性的鸟苷酸交换因子(GEFs),通过DH-PH结构域调节GTP与RhoA蛋白的结合,从而调节RhoA蛋白的活性,参与调控了多种生物学行为,如细胞骨架的构成,细胞的运动迁移,细胞与细胞之间以及细胞与基质之间的粘附等等。有研究表明,Net1蛋白激活RhoA蛋白可以促进胃癌细胞的侵袭转移能力。在稳定状态下,由于核定位信号(NLS)的存在,Net1蛋白定位于细胞核。缺失核定位信号(NLS)可以使Net1蛋白重新分布于细胞浆中,并且活化RhoA蛋白,促进肌动蛋白聚合形成张力丝。另一方面Net1蛋白可以通过C端的PDZ结合基序和肿瘤抑制蛋白D1g蛋白家族成员结合,使其进入细胞核内,从而发挥抑瘤作用。Net1蛋白重新定位于细胞浆激活RhoA蛋白是Net1蛋白转化细胞的关键步骤。Net1蛋白的生物信息学特征和研究结果提示Net1基因可能在肿瘤的发生发展中发挥了重要作用。本研究通过大样本检测Net1蛋白在肝细胞癌患者中的表达水平,并分析其和预后的关系；进一步通过分子生物学的实验研究其具体分子机制；从而初步阐明Net1蛋白在肝细胞癌发生发展过程中的机制及其意义。方法：采用免疫组织化学染色检测368例HCC及距肿瘤边缘至少2cm的癌旁肝组织(癌组织均经病理证实无癌细胞)样本中Net1的表达水平。通过Kaplan-Meier生存曲线和多元变量Cox回归统计学方法分析其和预后的关系。进一步使用RNA干扰技术,干扰高转移肝癌细胞株MHCC-97H的Net1蛋白的表达；运用生长曲线、MTT实验、划痕实验、基质胶侵袭实验以及裸鼠转移模型实验研究Net1蛋白对高转移肝癌细胞生物学行为的影响。最后采用酵母双杂交系统筛选Net1交互作用蛋白；并用免疫共沉淀的方法明确了交互作用的具体机制和意义。结果：在368例HCC组织中Net1呈高表达并且表达的阳性率达到86.7%(319/368),与Edmondson-Steiner分级(P=0.02)以及TNM分期(P=0.01)呈正相关。Net1表达水平与肝内转移(P=0.008)和门静脉侵润(P=0.007)呈正相关。高表达Net1的HCC病人比具有低表达Net1或表达缺失Net1(分别为P=0.001和P=0.002)的病人具有更短的无病存活期或者总存活期。多元Cox回归分析表明Net1蛋白表达(相对危险性RR,5.8；P=0.01)对HCC病人是一个独立的预后预测因素。由于Net1在肝细胞癌病人中为高表达,并且HCC组织中的Net1表达水平跟肝内转移(P=0.008)和门静脉侵润(P=0.007)呈正相关。提示高表达的Net1蛋白水平促进了肝癌细胞向体内的侵袭转移。为了证实这一假说,我们使用RNA干扰技术,干扰高转移肝癌细胞株MHCC-97H的Net1蛋白的表达。生长曲线和MTT实验表明,下调Net1蛋白的表达水平对MHCC-97H细胞的增殖能力没用明显作用；划痕实验,基质胶侵袭实验以及裸鼠转移模型实验表明,下调Net1蛋白的表达水平能够明显抑制MHCC-97H细胞的运动和侵袭转移能力。采用了酵母双杂交系统筛选Net1交互作用蛋白。筛选出Merlin和Net1蛋白具有交互作用。采用特异性酵母双杂交以及免疫共沉淀技术进一步证实了Net1与Merlin之间的交互作用。Merlin是NF2基因的编码产物,NF2基因是脑瘤的一个著名的抑瘤基因,在多种脑肿瘤里表达缺失。我们的研究发现,将NF2基因转染进肝癌细胞系MHCC-97H中,可以明显下调Net1蛋白的表达；并且随着NF2基因产物Merlin的表达依次增高,Net1的表达则依次降低。进一步研究发现,Merlin可以促进Net1在细胞内的泛素化。将pCMV-NF2质粒转染进MHCC-97H细胞,然后用不同浓度的MG132处理,裂解细胞,用Net1抗体进行免疫沉淀,再用泛素抗体进行Western Blot检测。结果表明,Net1被泛素化,并且随着MG132浓度的增高,泛素化的Net1也依次增高。同样的,将pCMV-NF2质粒(2μg)转染进MHCC-97H细胞,然后用不同浓度的MG132处理15小时,裂解细胞,用泛素亲和琼脂糖珠子免疫沉淀,再用Net1抗体进行Western Blot检测。结果同样显示：Net1被泛素化,并且随着MG132浓度的增高,泛素化的Net1也依次增高。另外将不同量的pCMV-NF2质粒分别转染进MHCC-97H细胞,逐渐增加Merlin的表达量,各组用相同浓度的MG132 (5μM)处理15小时,裂解细胞,用Net1抗体进行免疫沉淀,再用泛素抗体进行Western Blot检测,随着Merlin的表达的增加,可以逐步增加Net1蛋白的泛素化。这些结果说明了在细胞内Merlin可以促进Net1蛋白的泛素化,并使其通过蛋白酶体降解。结论：本研究通过检测肝细胞癌患者中的Net1蛋白表达水平,明确了在肝癌患者中Net1蛋白表达上调并且和转移及预后密切相关；下调Net1蛋白的表达水平可以明显抑制MHCC-97H细胞的运动以及侵袭转移能力；筛选并证实了Merlin和Net1蛋白发生交互作用；在细胞内Merlin可以促进Net1蛋白的泛素化,并使其通过蛋白酶体降解。更多还原

【Abstract】 Objective:Hepatocellular carcinoma (HCC), one of the most common fatal malignancies in China and many other countries in Asia and Africa, is the second cause of cancer mortality in China. To date, many studies have indicated many aberrantly expressed genes in HCC, but the molecular factors that can help to identify early tumor recurrence and serve as potential therapeutic target remain limited. Neuroepithelial cell transforming gene 1 (Netl) was originally identified as an oncogene in neuroepithelial cells and the mutant type could transform NIH3T3 cells. Netl is a RhoA specific guanine nucleotide exchange factor (GEF) which catalyze the exchange of GDP for GTP in RhoA protein. Rho GTPases control many aspects of cell behavior, such as the organization of the cytoskeleton, cell migration, cell-cell and cell-matrix adhesion, cell cycle progression, gene expression, and cell polarity. Recent research revealed that Netl mediated RhoA activation facilitates lysophosphatidic acid-induced cell migration and invasion in gastric cancer. At steady state, Netl localizes to the nucleus through the function of its NLS. Deletion of the N-terminal domain containing the NLS sequences redistributes Netl to the cytosol and promotes the formation of actin stress fibers, which is a consequence of RhoA activation. In addition, these studies demonstrated that the PDZ binding motif was essential for Netl-mediated transformation of NIH 3T3 cells. Netl interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP 102, and PSD95. The interaction between Netl and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. The oncogenic mutant of Netl is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. The ability of oncogenic Netl to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. To investigate its prognostic significance in HCC, which currently is unknown, we examined the correlation between Netl expression and prognosis in patients with HCC. Furthermore, we used many molecular biology experiments to prove and reveal the molecular mechanism of Netl in HCC.Method:Using immunohistochemical staining, and 368 pairs of HCC of at least 2cm away from the edge of the tumor adjacent liver tissue (cancer were pathologically confirmed non-cancer) samples in the expression level of Netl. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of HCC. RNA interference technology was used to knock down the expression level of the Netl protein in MHCC-97H cells. Growth curve and MTT were used to test the cell proliferation ability. Matrigel migration assay, scraping test and nude mice metastatic model were used to test the invasion and mobility of MHCC-97H cells. Finally We used yeast two-hybrid system to screen the protein interacted with Netl.Result:Among 368 specimens of HCC tissues,the Netl is high expression and the positive rate of Netl protein expression was 86.7%(319/368), and the increased Netl expression was correlated significantly with high Edmondson-Steiner grade (P=0.02) and TNM stage (P=0.01).Netl expression is associated with proliferation, metastasis and clinical stages of human hepatocellular carcinoma (HCC). The expression level of Netl in HCC tissues was associated with intrahepatic metastasis (P=0.008) and portal vein infiltration (P=0.007). Patients with HCC who had moderate-strong Netl positive expression had either poorer disease-free survival or poorer overall survival than patients who had with negative-low positive Netl expression (P=0.001 and P=0.002, respectively). Multivariate Cox regression analysis revealed that Netl protein expression (relative risk RR,5.8; P=0.01) was an independent prognostic factor for patients with HCC. Net1 status may be a new predictor of survival for HCC patients and provides the rationale for developing a novel therapy of targeting Netl against this fatal malignancy.The expression level of Netl in HCC tissues was associated with intrahepatic metastasis (P=0.008) and portal vein infiltration (P=0.007) suggested Netl may be play a very important role in tumor metastasis in HCC. To test this hypothesis, we used RNA interference technology to knock down the expression level of the Netl protein in MHCC-97H cells. Growth curve and MTT test revealed down-regulated Netl didn’t inhibit the proliferation ability in MHCC-97H cells. Expression silence of Netl by RNAi could inhibit the invasion and mobility of MHCC-97H cells detected by matrigel migration assay, scraping test and nude mice metastatic model.We used yeast two-hybrid system to screen the protein interacted with Netl. The results indicated Merlin could interacte with Netl. This interaction between Merlin and Netl also been proved by specific yeast two-hybrid and co-immunoprecipitation. Merlin is a production of NF2 gene. Neurofibromatosis type 2 (NF2) is a predominantly inherited disorder characterized by the development of schwann cell tumors and other brain tumors. Mutations or the loss of heterozygosity of the NF2 locus has been detected in various tumors of the nervous system, such as schwannomas, meningiomas and ependymomas. We demonstrated that the overexpression of merlin decreased the protein level of Netl and Netl is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin in the presence of MG132, a proteasome inhibitor.Conclusion:Netl is significantly up-regulated in the human hepatocellular carcinoma and associated with tumor metastasis; Decreasing the expression level of Netl can reduce the ability of invasion and migration of MHCC-97H cells significantly. Merlin interacts with Netl and facilitates degradation of Netl protein through ubiqutin-proteasomes pathway in MHCC-97H cells.更多还原