The Role And Possible Mechanism Of Notch Signaling In The Development And Progression Of Hepatocellular Carcinoma

[Background]Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Hepatitis B virus (HBV) infection is the leading cause of HCC in China, while the molecular mechanisms of the carcinogenesis and progression of HCC have not been clarified. Notch signaling pathway is an evolutionarily highly conserved mechanism for cell-cell communication, which controls differentiation and proliferation, and plays an important role in embryogenesis and many types of cell fate determination. There are reports showing that Notch signaling is also involved in the development of some cancers. The deregulation of Notch receptors and ligands and the aberrant activation of Notch signaling are found in a series of malignant tumors. Until now, the role of Notch signaling in HCC and its underlying mechanism are still unknown.[Objectives]1. To investigate the expression of Notch receptors and ligands in HCC; 2. To examine the relationship of Notch signaling with HBx; 3. To study the role of Notch signaling in malicious biological behaviors of HCC and its possible mechanism.[Methods]1. The expression of Notch receptors and ligands in human primary HCC tissues was detected by immunochemical staining; 2. The mRNA and protein levels of Notch receptors and ligands in HepG2X cells were examined by RT-PCR and Western Blot; 3. The mediated signaling molecules between HBx and the regulated Notch molecules were analyzed by detecting the alteration of the Notch molecules in HepG2X cells after treated with specific inhibitors of Ras, MEK, PI3K and p38; 4. The co-localization and physical interaction of relevant Notch molecules with HBx were analyzed by confocal microscopy and co-immunoprecipitation assay; 5. The activation of Notch signaling by HBx was examined by dual-luciferase reporter assay; 6. Eukaryotic expression vector for ICN1 was stably transfected into HepG2 cells, and siRNA vector of Notch1 was stably transfected into SMMC7721 cells; these cells were screened and identified; 7. The cells in which the whole Notch signaling was suppressed were achieved by treating SMMC7721 and HepG2X cells with a specific inhibitor of Notch signaling; 8. MTT method was used to examined the growth of the above transfectants and the Notch signaling-suppressed cells; colony formation assays in plate and in soft agar were used to detect the colony formation and anchor-independent growth ability; tumorigenesis assay in nude mice was used to investigate the tumorigenesis ability; 9. Cell cycle of those transfectants and the Notch signaling-suppressed cells was examined by FCM analysis; Annexinâ…´/PI dual-staining, DAPI staining, transition electron microscopy and TUNEL staining were used to detect cell apoptosis; 10. Gene array was used to detect the regulated molecules by ectopic ICN1 expression in HepG2 cells.[Result]1. Notch1, Notch2, Notch4 and Jagged1 were deregulated in HCC compared with adjacent nontomor liver.The expression of Notch1, Notch2, Notch3, Notch4 and Jagged1, Delta4 in human primary HCC tissues was examined by immunochemical staining. It was found that all of the examined Notch receptors and ligands were expressed in the neoplastic cells of HCC tissues with different extensity and intensity. Notch1 was expressed mainly in cytoplasm (88.7%), and also in nucleus in a few cases (9.4%); Notch4 showed nearly equal expression in cytoplasm (67.9%) and nucleus (58.5%); Notch2, Notch3, Jagged1 and Delta4 were expressed only in cytoplasm, with positive rate of 26.4%, 52.8%, 79.2% and 67.9%, respectively. Compared with adjacent nontomor liver, the expression of Notch1 (cytoplasmic), Notch4 (nuclear) and Jagged1 was significantly up-regulated in HCC, while the expression of Notch2 was significantly down-regulated. About the relationship of Notch molecules with clinicopathological parameters, the levels of Notch2, Notch3, Jagged1 and Delta4 were significantly higher in old (at the age of more than 50 years old) than in younger patients (less than 50 years old); the expression of Notch1 (cytoplasmic), Notch2, Notch3, Jagged1 and Delta4 had significant differences among differentiation grade. Besides, the expression of Notch1 (cytoplasmic), Notch4 (nuclear) and Jagged1 was found to be closely related with HBx in HBV-associated HCC tissues, respectively. 2. Notch signaling was regulated by HBx in HCC.The relationship of Notch signaling with HBx was further investigated in vitro. It was found that, consistent with the above in vivo result, the protein levels of Notch1 (NTM1), Notch4 (ICN4) and Jagged1 were higher in HepG2X cells than in mock transfectant, and the mRNA level of Notch1 and Jagged1 were also higher in HepG2X cells. The protein level of Notch1 was decreased in HepG2X cells after treated with the specific inhibitor of Ras or p38 while not the inhibitor of MEK or PI3K, which showed that HBx regulated the expression of Notch1 through Ras-p38 pathway.Besides, Notch1 and Jagged1 were found to co-localize with HBx in the cytoplasm of neoplastic cells of both HBV-associated HCC tissues and HepG2X cells, and these two Notch molecules were also found to physically interact with HBx in HepG2X cells. Moreover, Notch signaling in HepG2 cells could be activated by HBx in a dose-dependent manner.3. Notch signaling contributed to the growth and proliferation of HCC cells by promoting cell cycle progression and reducing cell apoptosis.To investigate the effects of Notch signaling on HCC cells, firstly, the expression of Notch1 in five HCC cell lines was examined and it was found that the protein level of Notch1 was high in SMMC7721 while low in HepG2 cells. Then eukaryotic expression vector for ICN1 was stably transfected into HepG2 cells, and siRNA vector of Notch1 was constructed and stably transfected into SMMC7721 cells; these cells were screened and verified. After that, the growth of these transfectants was detected: ectopic expression of ICN1 in HepG2 cells led to enhanced ability of cell growth and proliferation, colony formation, anchor-independent growth and tumorigenesis in nude mice, and also promotion of G1-S progression of cell cycle and reduction of cell apoptosis; consistent with it, knockdown of Notch1 by RNAi technique in SMMC7721 cells resulted in decreased ability of cell survival and proliferation, blockade of cell cycle progression, and increase of cell apoptosis. Besides, treatment with the specific inhibitor of Notch signaling to SMMC7721 and HepG2X cells also led to reduced ability of cell survival, blockade of cell cycle progression, and dramatic increase of cell apoptosis. Gene array showed that ectopic expression of ICN1 in HepG2 cells caused deregulation of a series of molecules affecting cell survival and migration.[Conclusions]1. Notch1, Notch2, Notch4 and Jagged1 are differently expressed in HCC compared with adjacent nontumor liver, which shows that these molecules might be involved in the carcinogenesis and progression of HCC. 2. HBx can regulate the expression and function of Notch1, Notch4 and Jagged1, and can thus activate Notch signaling. 3. Activation of Notch signaling can contribute to the growth and proliferation of HCC cells, promote cell cycle progression, and reduce cell apoptosis, while inhibition of it can suppress the survival of HCC cells, block cell cycle progression, and promote cell apoptosis. These effects are possibly related with some molecules affecting cell survial, which are regulated by Notch signaling. To sum up, the present work, for the first time, links Notch signaling to the development and progression of HCC.