MTBP Promotes The Invasion And Metastasis Of Hepatocellular Carcinoma By Enhancing The MDM2-mediated Degradation Of E-cadherin

Background and objective:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) in the current cancer mortality is in second place, and surgical resection is regarded as the main treatments for HCC. However, most patients initially are diagnosed in the middle and late stage, when lost the chance of radical surgery, only less than 20% of patients can be timely radical surgical resection. This is mainly because of the high degree of malignancy of HCC cells, the occurrence of metastasis earlily and rapid invasive growth. Thus, it is a key problem to investigate the molecular mechanisms involved in HCC invasion and metastasis and look for effective prevention targets in research of the treatment of HCC.The latest researches report MTBP can affect the growth of tumor in all aspects, including cell proliferation, migration, invasion, apoptosis and multidrug resistance gene expression. MTBP in tumor development plays an important role, but its effect on hepatocellular carcinoma (HCC) is unclear. We first found that the high expression of MTBP protein in hepatocellular carcinoma patients correlated with distant metastasis and poor prognosis. The expression of MTBP in metastatic cell lines increased in comparison with the non metastatic cell line. Consistent with these findings, enhanced the expression of MTBP promote the invasion and metastasis ability of HCCs in vitro and in vivo, and MTBP with small interfering RNA knockdown results in reduced migration and invasion of hepatocellular carcinoma. In tumor progression, tumor cells acquire mesenchymal marker expression and lose the expression of epithelial marker, resulting in epithelial mesenchymal transition (EMT) and subsequent tumor metastasis. The downregulation of E-cadherin (E-cadherin) is the hallmark of the EMT and is associated with the development of malignant epithelial carcinoma. Our preliminary screening shows that decreased MTBP expression resulted in increased expression of E-cadherin in hepatoma cells by gene chip. Hepatocellular carcinoma is a tumor with strong invasion and metastasis ability, so we speculated that MTBP may influence the invasion and metastasis of hepatocellular carcinoma cells through regulating the expression of E-cadherin. This study intends to detect the expression of MTBP in hepatocellular carcinoma and analyze the correlation between the expression of MTBP and the survival rate of HCCs. Secondly, MTBP expression effect on biological characteristics of invasion and the migration of HCCs by regulation of E-cadherin in vitro and in vivo. Finally, to investigate the molecular biological mechanism of MTBP regulation of E-cadherin.Method:l.By using qRT-PCR, Western blot and immunohistochemical method, Exploring MTBP expression in HCC tissue and cancer adjacent tissues of 120 cases and analyzing the relationship between MTBP expression and overall survival rates in 120 patients or patients in HCC recurrence by Kaplan-Meier analysis;2. Change the expression of MTBP in HCCs to detect the changes of the tumor invasion and metastasis by transwell, wound-healing assay and tumor formation in nude mice;3. Detection of the expression of E-cadherin protein by changing the expression of MTBP in HCCs through Western blot. Exploring the MTBP and E-cadherin protein expression levels in hepatocellular carcinoma tissues and para carcinoma tissue and analyze the correlation between them statistically;4. To determine the interaction of MDM2 and Ub by Western blot, CO immunoprecipitation, confocal laserand and how MTBP regulates MDM2 ubiquitination and thus change the expression of E-cadherin protein affect the ability of metastasis and invasion of HCC cells.Results:1. qRT-PCR and Western blot were applied to 120 HCC tissue samples and the corresponding adjacent tissues, the expression of MTBP in hepatocellular carcinoma tissues was significantly higher than that in paracancerous tissues.84 (70.0%) out of 120 HCC specimens showed high MTBP expression, whereas only 12 (10%) out of 120 normal liver tissue specimens showed high MTBP expression.The results of immunohistochemical staining also reveal that the expression of MTBP protein in tumor tissues was significantly higher than that of the corresponding para carcinoma tissues. And Our results indicate that overall survival rate in the patients with MTBP overexpression was significantly lower than that with low MTBP expression by Kaplan-Meier analysis.2. Western blot, Transwell and tumor formation in nude mice as experimental method confirm that enhanced expression of MTBP in HCC cells can promote HGC cell the invasion and metastasis whereas decreased expression of MTBP can reduce HCC cells the invasion and metastasis both in vitro and in vivo.3. MTBP over expression decreases E-cadherin expression in HCC cells, whereas MTBP low expression increases E-cadherin expression. MTBP and E-cadherin protein expression are inversely correlated in HCC tissues and para cancerous tissue by Western blot.4.By CO immunoprecipitation and confocal laser to determine the interaction between MDM2 and Ub, and confirm that the expression of MTBP can regulate MDM2 ubiquitination to inhibit E-cadherin protein to promote invasion and metastasis of HCCs by western blot and CO-IP.Conclusion:MTBP regulate the ubiquitin degradation of MDM2 protein induce the inhibition of E-cadherin gene expression and promote the invasion and metastasis of hepatocellular carcinoma. Our findings not only provide a new theoretical foundation for the mechanism of invasion and metastasis of hepatocellular carcinoma, but also suggest a new research direction for the prevention and treatment of HCC.