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。
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.
引文
[1]Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis:from genes to environment. Nat Rev Cancer.2006;6(9):674-87.
[2]Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet.2002;31(4):339-46.
[3]Gish RG, Porta C, Lazar L, et al. Phase Ⅲ randomized controlled trial comparing the survival of patients with unresectable hepatocellular carcinoma treated with nolatrexed or doxorubicin. J Clin Oncol.2007;25(21):3069-75.
[4]Chan AM, Takai S, Yamada K, Miki T. Isolation of a novel oncogene, NET1, from neuroepithelioma cells by expression cDNA cloning. Oncogene. 1996;12(6):1259-66.
[5]Garcia-Mata R, Dubash AD, Sharek L, Carr HS, Frost JA, Burridge K. The nuclear RhoA exchange factor Net1 interacts with proteins of the Dlg family, affects their localization, and influences their tumor suppressor activity. Molecular and Cellular Biology.2007;27(24):8683.
[6]Symons M, Rusk N. Control of vesicular trafficking by Rho GTPases. Curr Biol.2003;13(10):R409-18.
[7]Rossman KL, Der CJ, Sondek J. GEF means go:turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6(2):167-80.
[8]Van Aelst L, D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev.1997; 11 (18):2295-322.
[9]Burridge K, Wennerberg K. Rho and Rac take center stage. Cell. 2004;116(2):167-79.
[10]Hall A. Rho GTPases and the control of cell behaviour. Biochem Soc Trans. 2005;33(Pt5):891-5.
[11]Schmidt A, Hall A. The Rho Exchange Factor Net 1 Is Regulated by Nuclear Sequestration. Journal of Biological Chemistry.2002;277(17):14581-8.
[12]Alberts AS, Treisman R. Activation of RhoA and SAPK/JNK signalling pathways by the RhoA-specific exchange factor mNET1. EMBO J. 1998;17(14):4075-85.
[13]Carr HS, Cai C, Keinanen K, Frost JA. Interaction of the RhoA Exchange Factor Netl with Discs Large Homolog 1 Protects It from Proteasome-mediated Degradation and Potentiates Netl Activity. Journal of Biological Chemistry.2009;284(36):24269.
[14]Mackay DJ, Hall A. Rho GTPases. JBiol Chem.1998;273(33):20685-8.
[15]Nobes CD, Hall A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. Journal of Cell Biology.1999; 144(6):1235.
[16]Meriane M, Mary S, Comunale F, Vignal E, Fort P, Gauthier-Rouviere C. Cdc 42 Hs and Rac 1 GTPases Induce the Collapse of the Vimentin Intermediate Filament Network. Journal of Biological Chemistry.2000;275(42):33046-52.
[17]Kaibuchi K, Kuroda S, Amano M. Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annual review of biochemistry.1999;68(1):459-86.
[18]Bar-Sagi D, Hall A. Ras and Rho GTPases a family reunion. Cell. 2000;103(2):227-38.
[19]Ridley AJ. Rho family proteins:coordinating cell responses. Trends in Cell Biology.2001;11(12):471-7.
[20]Hall A. G proteins and small GTPases:distant relatives keep in touch. Science (Washington, D C).1998;280(5372):2074-5.
[21]Zalcman G, Closson V, Camonis J, et al. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a non-cytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. Journal of Biological Chemistry.1996;271(48):30366.
[22]Murray D, Horgan G, Macmathuna P, Doran P. NET1-mediated RhoA activation facilitates lysophosphatidic acid-induced cell migration and invasion in gastric cancer. British Journal of Cancer.2008;99(8):1322-9.
[23]高进.中国癌症侵袭与转移研究的回顾与展望.中华肿瘤杂志.2001；23(265-8.
[24]曾益新主编.肿瘤学.北京：人民卫生出版社1999,174-175.
[25]Ishak KG, Anthony PP, Sobin LH. Histological Typing of Tumours of the Liver (WHO. World Health Organization. International Histological Classification of Tumours).2 ed. Berlin:Springer 1994.
[26]Wittekind C. Pitfalls in the classification of liver tumors. Pathologe. 2006;27(4):289-93.
[27]Gao Q, Qiu SJ, Fan J, et al. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol.2007;25(18):2586-93.
[28]Zhu XD, Zhang JB, Zhuang PY, et al. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. Journal of Clinical Oncology.2008;26(16):2707.
[29]Luo J, Isaacs WB, Trent JM, Duggan DJ. Looking beyond morphology:cancer gene expression profiling using DNA microarrays. Cancer investigation. 2003;21(6):937-49.
[30]DeRisi J, Penland L, Brown PO, et al. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nature genetics. 1996;14(4):457-60.
[31]Velculescu VE, Zhang L, Vogelstein B, Kinzler KW. Serial analysis of gene expression. Science.1995;270(5235):484-7.
[32]Sallinen SL, Sallinen PK, Haapasalo HK, et al. Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques. Cancer research.2000;60(23):6617.
[33]Moch H, Schraml P, Bubendorf L, et al. High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. American Journal of Pathology.1999;154(4):981.
[34]Bubendorf L, Kononen J, Koivisto P, et al. Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res.1999;59(4):803-6.
[35]Mohr S, Leikauf GD, Keith G, Rihn BH. Microarrays as cancer keys:an array of possibilities. Journal of Clinical Oncology.2002;20(14):3165.
[36]Bremnes RM, Veve R, Gabrielson E, et al. High-throughput tissue microarray analysis used to evaluate biology and prognostic significance of the E-cadherin pathway in non-small-cell lung cancer. Journal of Clinical Oncology.2002;20(10):2417.
[37]Bubendorf L, Nocito A, Moch H, Sauter G. Tissue microarray TMA technology:miniaturized pathology archives for high-throughput in situ studies. The Journal of Pathology.2001;195(1):72-9.
[38]Richter J, Wagner U, Kononen J, et al. High-throughput tissue microarray analysis of cyclin E gene amplification and overexpression in urinary bladder cancer. American Journal of Pathology.2000;157(3):787.
[39]Kononen J, Bubendorf L, Kallionimeni A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Medicine. 1998;4(7):844-7.
[40]Swierczynski SL, Maitra A, Abraham SC, et al. Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. Human pathology.2004;35(3):357-66.
[41]Hewitt SM. Design, Construction, and Use of Tissue Microarrays. Protein Arrays:Methods and Protocols 2004:61-72.
[42]Skotheim RI, Abeler VM, Nesland JM, et al. Candidate genes for testicular cancer evaluated by in situ protein expression analyses on tissue microarrays. Neoplasia 2003;5(5):397.
[43]Henshall S. Tissue microarrays. J Mammary Gland Biol Neoplasia. 2003;8(3):347-58.
[44]Kallioniemi OP, Wagner U, Kononen J, Sauter G. Tissue microarray technology for high-throughput molecular profiling of cancer. Human molecular genetics.2001;10(7):657.
[45]Korabiowska M, Bauer H, Quentin T, Stachura J, Cordon-Cardo C, Brinck U. Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas:correlation with immunohistochemical analysis. Human pathology.2004;35(2):210-6.
[46]Hidalgo A, Pina P, Guerrero G, Lazos M, Salcedo M. A simple method for the construction of small format tissue arrays. British Medical Journal. 2003;56(2):144.
[47]Mucci NR, Akdas G, Manely S, Rubin MA. Neuroendocrine expression in metastaticprostate cancer:Evaluation of high throughput tissue microarrays to detect heterogeneous protein expression. Human pathology. 2000;31(4):406-14.
[48]Packeisen J, Korsching E, Herbst H, Boecker W, Buerger H. Demystified.. tissue microarray technology. Molecular pathology.2003;56(4):198.
[49]Mengel M, Kreipe H, von Wasielewski R. Rapid and large-scale transition of new tumor biomarkers to clinical biopsy material by innovative tissue microarray systems. Applied Immunohistochemistry & Molecular Morphology. 2003;11(3):261.
[50]Gillespie JW, Best CJM, Bichsel VE, et al. Evaluation of non-formalin tissue fixation for molecular profiling studies. American Journal of Pathology. 2002;160(2):449.
[51]Lewis F, Maughan NJ, Smith V, Hillan K, Quirke P. Unlocking the archive-gene expression in paraffin-embedded tissue. The Journal of Pathology.2001;195(1):66-71.
[52]Frantz GD, Pham TQ, Peale FV, Hillan KJ. Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays. The Journal of Pathology.2001;195(1):87-96.
[53]Andersen CL, Monni O, Wagner U, et al. High-throughput copy number analysis of 17q23 in 3520 tissue specimens by fluorescence in situ hybridization to tissue microarrays. American Journal of Pathology. 2002;161(1):73.
[54]Tynninen O, Paetau A, Von Boguslawski K, J skel inen J, Aronen HJ, Paavonen T. p53 expression in tissue microarray of primary and recurrent gliomas. Brain Pathol.2000;10(4):575-6.
[55]Barlund M, Monni O, Kononen J, et al. Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer research. 2000;60(19):5340.
[56]Schraml P, Kononen J, Bubendorf L, et al. Tissue microarrays for gene amplification surveys in many different tumor types. Clinical Cancer Research.1999;5(8):1966.
[57]Bubendorf L, Kolmer M, Kononen J, et al. Hormone therapy failure in human prostate cancer:analysis by complementary DNA and tissue microarrays. JNCI Journal of the National Cancer Institute.1999;91(20):1758.
[58]Wang L, Deavers MT, Malpica A, Silva EG, Liu J. Tissue macroarray:a simple and cost-effective method for high-throughput studies. Applied Immunohistochemistry & Molecular Morphology.2003;11(2):174.
[59]Matysiak BE, Brodzeller T, Buck S, et al. Simple, inexpensive method for automating tissue microarray production provides enhanced microarray reproducibility. Applied Immunohistochemistry & Molecular Morphology. 2003;11(3):269.
[60]Zhang DH, Salto-Tellez M, Putti TC, Do E, Koay ESC. Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer. Modern Pathology.2003;16(1):79-85.
[61]Hedenfalk I, Duggan D, Chen Y, et al. Gene-expression profiles in hereditary breast cancer. New England Journal of Medicine.2001;344(8):539.
[62]Gillett CE, Springall RJ, Barnes DM, Hanby AM. Multiple tissue core arrays in histopathology research:a validation study. The Journal of Pathology. 2000;192(4):549-53.
[63]Camp RL, Charette LA, Rimm DL. Validation of tissue microarray technology in breast carcinoma. Laboratory Investigation.2000;80(12):1943-9.
[64]Liu CL, Prapong W, Natkunam Y, et al. Software tools for high-throughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays. American Journal of Pathology.2002;161(5):1557.
[65]吴阶平,裘法祖,黄家驷.外科学.6 ed.北京：人民卫生出版社2004.
[66]Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature.1998;391(6669):806-11.
[67]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature.2001;411(6836):494-8.
[68]Li Y, Tang ZY, Ye SL, et al. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol.2001;7(5):630-6.
[69]Tian J, Tang ZY, Ye SL, et al. New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer.1999;81(5):814-21.
[70]Sun FX, Tang ZY, Lui KD, et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer.1996;66(2):239-43.
[71]Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990;50(19):6130-8.
[72]卢大用,曹静懿.肿瘤转移的实验病理学研究.河南医学研究.2000；9(003)：282-5.
[73]Torimura T, Ueno T, Kin M, et al. Coordinated expression of integrin a6β1 and laminin in hepatocellular carcinoma. Human pathology. 1997;28(10):1131-8.
[74]Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of the National Academy of Sciences.2002;99(9):6047.
[75]Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proceedings of the National Academy of Sciences.2002;99(8):5515.
[76]Paul CP, Good PD, Winer I, Engelke DR. Effective expression of small interfering RNA in human cells. Nature Biotechnology.2002;20(5):505-8.
[77]Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol.2002;20(5):497-500.
[78]Hannon GJ. RNA interference. Nature.2002;418(6894):244-51.
[79]Brown D, Jarvis R, Pallotta V, Byrom M, Ford L. RNA interference in mammalian cell culture:design, execution, and analysis of the siRNA effect. Ambion TechNotes.2002;9(1):3-5.
[80]Bracke ME, Boterberg T, Bruyneel EA. Collagen invasion assay. In:Brooks S, Schumacher U, eds. Metastasis Research Protocols. Totowa. New Jersey: Humana Press 2001:81-90.
[81]DMS H, SA B. In vitro invasion assay using Matrigel. In:Brooks S, Schumacher U, eds. Metastasis Research Protocols. Totowa. New Jersey: Humana Press 2001.
[82]高进.癌的侵袭与转移---基础研究与临床.北京：北京医科大学、中国协和医科大学联合出版社 1996.
[83]Tang ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma. Journal of cancer research and clinical oncology. 2004;130(4):187-96.
[84]Leyden J, Murray D, Moss A, et al. Netl and Myeov:computationally identified mediators of gastric cancer. Br J Cancer.2006;94(8):1204-12.
[85]Ito T, Ota K, Kubota H, et al. Roles for the two-hybrid system in exploration of the yeast protein interactome. Molecular & Cellular Proteomics. 2002;1(8):561.
[86]Gietz RD, Triggs-Raine B, Robbins A, Graham KC, Woods RA. Identification of proteins that interact with a protein of interest:applications of the yeast two-hybrid system. Molecular and cellular biochemistry.1997; 172(1):67-79.
[87]Fields S, Song O. A novel genetic system to detect protein protein interactions. Nature.1989;340(6230):245-6.
[88]卞留贯,孙青芳,罗其中,赵卫国,沈建康.Ⅱ型多发神经纤维瘤病.中国临床神经外科杂志.2004；9(002)：159-60.
[89]Schulze KMM, Hanemann CO, Muller HW, Hanenberg H. Transduction of wild-type merlin into human schwannoma cells decreases schwannoma cell growth and induces apoptosis. Human molecular genetics.2002;11(1):69.
[90]Shaw RJ, Paez JG, Curto M, et al. The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling. Developmental Cell.2001;1(1):63-72.
[91]Shaw RJ, McClatchey AI, Jacks T. Regulation of the neurofibromatosis type 2 tumor suppressor protein, merlin, by adhesion and growth arrest stimuli. Journal of Biological Chemistry.1998;273(13):7757.
[92]Morrison H, Sherman LS, Legg J, et al. The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44. Genes & development.2001;15(8):968.
[93]Aplin AE, Howe A, Alahari SK, Juliano RL. Signal transduction and signal modulation by cell adhesion receptors:the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacological reviews.1998;50(2):197-264.
[94]Bashour AM, Meng JJ, Ip W, MacCollin M, Ratner N. The neurofibromatosis type 2 gene product, merlin, reverses the F-actin cytoskeletal defects in primary human Schwannoma cells. Molecular and Cellular Biology. 2002;22(4):1150.
[95]Gutmann DH, Hirbe AC, Haipek CA. Functional analysis of neurofibromatosis 2 (NF2) missense mutations. Human molecular genetics. 2001;10(14):1519.
[96]Kim H, Kwak NJ, Lee JY, et al. Merlin neutralizes the inhibitory effect of Mdm2 on p53. Journal of Biological Chemistry.2004;279(9):7812.
[97]Lee JY, Kim H, Ryu CH, et al. Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity. Journal of Biological Chemistry.2004;279(29):30265.
[98]Lee JY, Moon HJ, Lee WK, et al. Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein. Oncogene. 2006;25(8):1143-52.
[99]Li W, You L, Cooper J, et al. Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus. Cell. 2010;140(4):477-90.
[100]Handley-Gearhart PM, Stephen AG, Trausch-Azar JS, Ciechanover A, Schwartz AL. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs. Journal of Biological Chemistry.1994;269(52):33171-8.
[101]Haas AL, Warms JV, Hershko A, Rose IA. Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. Journal of Biological Chemistry.1982;257(5):2543-8.
[102]Ciechanover A, Elias S, Heller H, Hershko A. " Covalent affinity" purification of ubiquitin-activating enzyme. Journal of Biological Chemistry. 1982;257(5):2537-42.
[103]Kumar S, Kao WH, Howley PM. Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity. J Biol Chem. 1997;272(21):13548-54.
[104]Cenci G, Rawson RB, Belloni G, et al. UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev.1997;11(7):863-75.
[105]Bailly V, Prakash S, Prakash L. Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein. Mol Cell Biol.1997;17(8):4536-43.
[106]Tachibana M, Iwata N, Watanabe A. Assignment of gene for a ubiquitin-conjugation enzyme (UBE2I) to human chromosome band 16p13. 13 by in situ hybridization. Cytogenet Cell Genet.1996;75(222-3.
[107]Nuber U, Schwarz S, Kaiser P, Schneider R, Scheffner M. Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5. J Biol Chem. 1996;271(5):2795-800.
[108]Hochstrasser M. Ubiquitin-dependent protein degradation. Annu Rev Genet. 1996;30(405-39.
[109]Jensen JP, Bates PW, Yang M, Vierstra RD, Weissman AM. Identification of a family of closely related human ubiquitin conjugating enzymes. J Biol Chem. 1995;270(51):30408-14.
[110]Jensen JP, Bates PW, Yang M, Vierstra RD, Weissman AM. Identification of a family of closely related human ubiquitin conjugating enzymes. Journal of Biological Chemistry.1995;270(51):30408.
[111]Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26(179-207.
[112]Dohmen RJ, Madura K, Bartel B, Varshavsky A. The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. Proc Natl Acad Sci USA. 1991;88(16):7351-5.
[113]Reiss Y, Heller H, Hershko A. Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein. J Biol Chem..1989;264(18):10378-83.
[114]Tasaki T, Kwon YT. The mammalian N-end rule pathway:new insights into its components and physiological roles. Trends in Biochemical Sciences. 2007;32(11):520-8.
[115]Mogk A, Schmidt R, Bukau B. The N-end rule pathway for regulated proteolysis:prokaryotic and eukaryotic strategies. Trends Cell Biol. 2007;17(4):165-72.
[116]Yang Y, Lorick KL, Jensen JP, Weissman AM. Expression and evaluation of RING finger proteins. Methods Enzymol.2005;398(103-12.
[117]Hatakeyama S, Nakayama KI. U-box proteins as a new family of ubiquitin ligases. Biochem Biophys Res Commun.2003;302(4):635-45.
[118]Ditzel M, Wilson R, Tenev T, et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat Cell Biol.2003;5(5):467-73.
[119]Jackson PK, Eldridge AG. The SCF ubiquitin ligase:an extended look. Mol Cell.2002;9(5):923-5.
[120]Swaroop M, Gosink M, Sun Y. SAG/ROC2/Rbx2/Hrt2, a component of SCF E3 ubiquitin ligase:genomic structure, a splicing variant, and two family pseudogenes. DNA Cell Biol.2001;20(7):425-34.
[121]Jiang J, Ballinger CA, Wu Y, et al. CHIP is a U-box-dependent E3 ubiquitin ligase:identification of Hsc70 as a target for ubiquitylation. J Biol Chem. 2001;276(46):42938-44.
[122]倪晓光,赵平.泛素-蛋白酶体途径的组成和功能.生理科学进展.2006；37(003)：255-8.
[123]Mukhopadhyay D, Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science.2007;315(5809):201.
[1]Sun CX, Robb VA, Gutmann DH. Protein 4.1 tumor suppressors:getting a FERM grip on growth regulation. Journal of cell science.2002;115(21):3991.
[2]Bretscher A, Edwards K, Fehon RG. ERM proteins and merlin:integrators at the cell cortex. Nature Reviews Molecular Cell Biology.2002;3(8):586-99.
[3]Gutmann DH, Hirbe AC, Haipek CA. Functional analysis of neurofibromatosis 2 (NF2) missense mutations. Human molecular genetics. 2001;10(14):1519.
[4]Li Q, Nance MR, Kulikauskas R, et al. Self-masking in an Intact ERM-merlin Protein:An Active Role for the Central [alpha]-Helical Domain. Journal of molecular biology.2007;365(5):1446-59.
[5]Terawaki S, Maesaki R, Hakoshima T. Structural basis for NHERF recognition by ERM proteins. Structure.2006; 14(4):777-89.
[6]Shimizu T, Seto A, Maita N, Hamada K, Tsukita S, Hakoshima T. Structural basis for neurofibromatosis type 2. Journal of Biological Chemistry. 2002;277(12):10332.
[7]Kang BS, Cooper DR, Devedjiev Y, Derewenda U, Derewenda ZS. The structure of the FERM domain of merlin, the neurofibromatosis type 2 gene product. Acta Crystallographica Section D:Biological Crystallography. 2002;58(3):381-91.
[8]Pearson MA, Reczek D, Bretscher A, Karplus PA. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell.2000;101(3):259-70.
[9]Weinman EJ, Hall RA, Friedman PA, Liu-Chen LY, Shenolikar S. The association of NHERF adaptor proteins with g protein-coupled receptors and receptor tyrosine kinases. Annu Rev Physiol.2006;68(491-505.
[10]Kissil JL, Wilker EW, Johnson KC, Eckman MS, Yaffe MB, Jacks T. Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pakl. Molecular cell.2003;12(4):841-9.
[11]Maeda M, Matsui T, Imamura M, Tsukita S. Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with Ezrin/Radixin/Moesin proteins. Oncogene.1999;18(34):4788-97.
[12]LaJeunesse DR, McCartney BM, Fehon RG. Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization. Journal of Cell Biology.1998;141(7):1589.
[13]Takahashi K, Sasaki T, Mammoto A, et al. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. Journal of Biological Chemistry.1997;272(37):23371.
[14]Tsukita S, Yonemura S. ERM proteins:head-to-tail regulation of actin-plasma membrane interaction. Trends in Biochemical Sciences.1997;22(2):53-8.
[15]McClatchey AI, Giovannini M. Membrane organization and tumorigenesis-the NF2 tumor suppressor, Merlin. Genes & development. 2005; 19(19):2265.
[16]Xiao GH, Beeser A, Chernoff J, Testa JR. p21-activated kinase links Rac/Cdc42 signaling to merlin. Journal of Biological Chemistry. 2002;277(2):883.
[17]Kissil JL, Johnson KC, Eckman MS, Jacks T. Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization. Journal of Biological Chemistry.2002;277(12):10394.
[18]Okada T, Lopez-Lago M, Giancotti FG. Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane. Journal of Cell Biology.2005;171(2):361.
[19]Lallemand D, Manent J, Couvelard A, et al. Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas. Oncogene.2008.
[20]Lallemand D, Curto M, Saotome I, Giovannini M, McClatchey AI. NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions. Genes & development.2003; 17(9):1090.
[21]Lopez-Lago MA, Okada T, Murillo MM, Socci N, Giancotti FG. Loss of the Tumor Suppressor Gene NF2, Encoding Merlin, Constitutively Activates Integrin-Dependent mTORC1 Signaling. Molecular and Cellular Biology. 2009;29(15):4235.
[22]Kunda P, Pelling AE, Liu T, Baum B. Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis. Current Biology. 2008;18(2):91-101.
[23]Carreno S, Kouranti I, Glusman ES, Fuller MT, Echard A, Payre F. Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. Journal of Cell Biology.2008;180(4):739.
[24]Charras GT, Hu CK, Coughlin M, Mitchison TJ. Reassembly of contractile actin cortex in cell blebs. Journal of Cell Biology.2006;175(3):477.
[25]Cole BK, Curto M, Chan AW, McClatchey AI. Localization to the cortical cytoskeleton is necessary for Nf2/merlin-dependent epidermal growth factor receptor silencing. Molecular and Cellular Biology.2008;28(4):1274.
[26]Laulajainen M, Muranen T, Carpen O, Gr nholm M. Protein kinase A-mediated phosphorylation of the NF2 tumor suppressor protein merlin at serine 10 affects the actin cytoskeleton. Oncogene.2007;27(23):3233-43.
[27]James MF, Manchanda N, Gonzalez-Agosti C, Hartwig JH, Ramesh V. The neurofibromatosis 2 protein product merlin selectively binds F-actin but not G-actin, and stabilizes the filaments through a lateral association. Biochemical Journal.2001;356(Pt 2):377.
[28]Pelton PD, Sherman LS, Rizvi TA, et al. Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells. Oncogene.1998;17(17):2195.
[29]Tikoo A, Varga M, Ramesh V, Gusella J, Maruta H. An anti-Ras function of neurofibromatosis type 2 gene product (NF2/Merlin). Journal of Biological Chemistry.1994;269(38):23387.
[30]McClatchey AI, Fehon RG. Merlin and the ERM proteins-regulators of receptor distribution and signaling at the cell cortex. Trends in Cell Biology. 2009; 19(5):198-206.
[31]Manchanda N, Lyubimova A, Ho HYH, et al. The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function. Journal of Biological Chemistry. 2005;280(13):12517.
[32]Rangwala R, Banine F, Borg JP, Sherman LS. Erbin regulates mitogen-activated protein (MAP) kinase activation and MAP kinase-dependent interactions between Merlin and adherens junction protein complexes in Schwann cells. Journal of Biological Chemistry.2005; 280 (12):11790.
[33]Curto M, Cole BK, Lallemand D, Liu CH, McClatchey AI. Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. Journal of Cell Biology. 2007;177(5):893.
[34]von Zastrow M, Sorkin A. Signaling on the endocytic pathway. Current opinion in cell biology.2007;19(4):436-45.
[35]Lazar CS, Cresson CM, Lauffenburger DA, Gill GN. The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface. Molecular biology of the cell.2004;15(12):5470.
[36]Kolch W. Erbin:sorting out ErbB2 receptors or giving Ras a break? Science Signaling.2003;2003(199).
[37]Fraenzer JT, Pan H, Minimo Jr L, Smith GM, Knauer D, Hung G. Overexpression of the NF2 gene inhibits schwannoma cell proliferation through promoting PDGFR degradation. International journal of oncology. 2003;23(6):1493.
[38]Bovie C, Holden ST, Schroer A, Smith E, Trump D, Raymond FL. Neurofibromatosis 2 in a patient with a de novo balanced reciprocal translocation 46, X, t (X; 22)(p11.2; qll.2). Journal of medical genetics. 2003;40(9):682.
[39]Kim H, Kwak NJ, Lee JY, et al. Merlin neutralizes the inhibitory effect of Mdm2 on p53. Journal of Biological Chemistry.2004;279(9):7812.
[40]Lee JY, Kim H, Ryu CH, et al. Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity. Journal of Biological Chemistry.2004;279(29):30265.
[41]Lee JY, Moon HJ, Lee WK, et al. Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein. Oncogene. 2006;25(8):1143-52.
[42]Li W, You L, Cooper J, et al. Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus. Cell. 2010;140(4):477-90.