| 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.
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