The Mechanism Of Fbxw7 Involving In Hepatocellular Carcinoma Migration And Invasion

Object: Hepatocellular carcinoma(HCC) is one of the most common human cancers. The incidence and mortality was always been high in cancer-related diseases. In China, HCC is the second leading cause of cancer-related death. With the advances in medical technology, although the better diagnostic technique for liver cancer, and gradually standardized surgery, adjuvant chemotherapy, and the popularity of liver transplantation, the prognosis of HCC patients is still not optimistic. Because of the initially asymptomatic development and unpredictable tumor metastasis of HCC, most patients have a limited number of therapeutic options at the time of diagnosis. The problem of recurrence and drug resistance, The survival rate of liver cancer is still very low. Both patients and doctors are full of fear of HCC. The rapid development of medicine is inseparable from the continuous advancement of medical research. So far, scholars from all around the world have found a large number of molecules, pathways and mechanisms associated with the growth, proliferation, metastasis, and drug resistance of cancer cells. Results of scientific research gradually converted to clinic, such as molecular targeted therapy, stem cell therapy, etc. There are so many complicated mechanism with HCC and need us to find. More than 90% of cancer-related deaths are due to the metastasis. We must put the research of metastasis on the most important position.Ubiquitin-proteasome system(UPS) is an important system of post-translational modification of protein, involved in the regulation of cell cycle, genetic transcription, cell signal transduction, apoptosis and differentiation and development, etc. Fbxw7(F-box and WD repeat domain containing 7) is an F-box protein belongs to SCF(SKP1-CUL1-F-box protein) E3 ligase complex. Fbxw7 is responsible for transfering the ubiquitin molecule to the substrate, resulting in its recognition and degradation. It can identify a variety of oncoprotein and promote its ubiquitination and degradation. Therefore, Fbxw7 is an important tumor suppressor gene that plays a significant biological function. The mutation or decreased expression of Fbxw7 will cause a variety of tumors.In recent years, Fbxw7 become focus of the research. The research concentrated on the outcome of stem cells and cancer stem cells, tumor growth, invasion and resistance, etc. There are seveal researches of HCC, including the expression in the HCC tissues and teh proliferation of HCC cells.However, whether Fbxw7/Notch1 axis participates in the process of metastasis and invasion in HCC remains unclear. Therefore, we designed and implemented the following researches to verify whether Fbxw7 play a role in HCC metastasis. Methods:We first collected tissue samples of 102 patients with HCC. The tissues were paraffin-embedded and filleted. Then the immunohistochemical staining was used to observe Fbxw7 expression in hepatocellular carcinoma and para-carcinoma tissue. Then we analized the significance of Fbxw7 for HCC combined with the clinical pathological data. Then, with Real-time PCR and Western-blot were used to detect the expression of Fbxw7 m RNA and protein levels in HCC lines. Then we built a stable down-regulated/up-regulated Fbxw7 viral vector and transfected into HCC lines. With real-time PCR, Western-blot, ELISA, Transwell chamber experimental, nude mice models of lung metastasis, we detected the relevant data of metastasis with HCC cells and analyzed the mechanism. Results:(1) We examined Fbxw7 expression level using HCC tissue microarray and immunohistochemistry. Our data showed that Fbxw7 expression is significantly reduced in HCC compared with noncancerous tissues(P < 0.05). Fbxw7 levels were significantly associated with tumor differentiation(P = 0.013), the incidence of portal or hepatic venous invasion(P = 0.031), metastasis(P = 0.027), and AJCC cancer stage(P = 0.047). Then we observed a strong correlation between low Fbxw7 expression and a worse 5-year survival of HCC patients(P < 0.001). Furthermore, multivariate Cox regression analyses demonstrated that the Fbxw7 expression(P < 0.001) was an independent factor for the prediction of the overall survival of HCC patients.(2) We examined the m RNA and protein levels of Fbxw7 in different HCC cell lines(i.e., Hep G2, SMMC-7721 and MHCC97H) compared with the human liver non-tumor cell line HL-7702. The m RNA levels of Fbxw7 were significantly down-regulated in HCC cells compared with HL-7702 cells. The western-blot analysis also showed that the expression levels of Fbxw7 protein exhibited similar decreased tendencies in these HCC cell lines.(3) We established lentivirus-mediated stable KD-Fbxw7-G2 and OE-Fbxw7-97 H cells, and their respective negative controls(i.e., NC-Fbxw7-G2 and NC-Fbxw7-97 H cells).Lentivirus infection was highly efficient and did not perturb endogenous Fbxw7 expression in HCC cells.(4) Using the Transwell cell culture chambers and nude mice models of lung metastasis, the ability of invasion and migration of KD-Fbxw7-G2 cells were stronger than NC-Fbxw7-G2 cells. The ability of invasion and migration of OE-Fbxw7-97 H cells were weaker than NNC-Fbxw7-97 H cells.(5) Western-blot was used to detect the protein expression of activated Notch1 in KD-Fbxw7-G2 and OE-Fbxw7-97 H cells. Then we detected the activation of ERK1/2 and MMP-2, MMP-9 and u PA. We found that inhibition of Fbxw7 upregulated expression of N1 ICD, and activated ERK1/2, MMP-2, MMP-9 and u PA. Then we down-regulated the expression of Notch1 using the si RNA, with the Transwell cell culture chambers, si Notch1 decreased the number of KD-Fbxw7-G2 cells migrated and make the cell numbers almost return to the status of untransfected. And the results of invasion were the same as the migration parts. Conclusion:Our findings indicate that Fbxw7 can be used as a prognostic marker; it has an important role in HCC progression and inhibits HCC cells migration and invasion via Fbxw7/Notch1/ERK1/2 axis.