The Molecular Mechanism Of Antitumor And Antifibrogenic Efficacy Of BCL6B In Hepatocellular Carcinoma

At present, liver cancer is one of the most common malignant tumors and the second cause of death of malignant tumors in China. Hepatic inflammation and fibrogenesis are the major etiologic causes leading to liver cancer, which also brings heavy burden to the quality of life. The therapy to liver cancer, especially the hepatocellular carcinoma(HCC) with fibrosis, is still ineffective except the hepatectomy. Tumor biomarkers screening and gene therapy by genetic technology are the most highlighted research that are being actively studied. B cell CLL/lymphoma 6 member B (BCL6B) is a novel functional tumor suppressor, which is down-regulated or silenced in gastric cancer, but its exact function in liver cancer is still unclear. It was reported that the reduced expression of BCL6B in tumor is associated with the promoter hypermethylation status and the poorer survival in gastric cancer patients, implicating the correlation between BCL6B expression and oncogenesis.Part I Antitumor efficacy of BCL6B in hepatocellular carcinomaAimsTo identify the BCL6B expression in hepatocellular carcinoma, and to evaluate the role BCL6B plays in suppressing tumor development.MethodsBCL6B expression was studied by quantitative PCR, Western Blot(WB) and immunohistochemistry(IHC) in both liver cancer tissues and adjacent normal tissues. The overexpression BCL6B cell models were established by exogenously transfecting recombinant BCL6B lentivirus to HepG2 and SMMC-7721 cell lines. The biological function of BCL6B were determined by cell viability, colony formation, flow cytometry, Transwell assays and in vivo tumorigenicity assays. Transcriptomic microarray analysis was performed to explore the mechanisms in which BCL6B confers protection from tumorigenesis.ResultsBCL6B expression was down-regulated or silenced in all seven HCC cell lines but readily expressed in normal hepatic cell line. Re-expression of BCL6B in HepG2 and SMMC-7721 cell lines suppressed cell viability and colony formation, induced apoptosis and cell-cycle arrest, inhibited the migration and invasion of HCC in vivo. Moreover, up-regulation of BCL6B also significantly suppressed the growth of subcutaneous tumors in nude mice in vivo. In addition, transcriptomic microarray revealed that BCL6B may suppress the tumor development through several key signal pathways.Quantitative PCR demonstrated that the BCL6B expression was reduced in HCC tissues compared to adjacent normal tissues in mRNA level, following confirmed by WB and IHC in protein level. Low BCL6B expression in tumors was correlated with shorter overall survival in patients, and multivariate Cox regression analysis revealed that BCL6B expression was an independent prognostic factor for human HCC patients. Moreover, a positive correlation between BCL6B expression and hepatic cirrhosis was found in an analysis of HCC clinicopathological characteristics.ConclusionThe reduced expression of BCL6B in HCC tissues was associated with absence of cirrhosis and poorer survival. BCL6B may play a key role in HCC proliferation, apoptosis and migration through several key signal pathways.Part â…¡ Antifibrogenic efficacy of BCL6B in damaged liverAims To identify the BCL6B expression in hepatic fibrogenic tissues, and to evaluate the roles BCL6B plays in hepatic fibrogenesis.MethodsRats were treated with diethylnitrosamine (DEN) or carbon tetrachloride (CCl4) to establish acute or chronic liver injury models, respectively. To establish liver-specific BCL6B transgenic(Tg) rat models, lentivirus of BCL6B or GFP were injected through the portal vein. Moreover, adenovirus of BCL6B or GFP were injected through tail vein to make BCL6B knockdown in rat livers. To determine the function of BCL6B in hepatic fibrogenesis, the expression level of some inflammation factors and fibrogenic biomarkers had been detected in this study.ResultsBCL6B expression was increased in hepatic tissues of chronic liver disease and rat fibrotic liver sample response to liver injury. Re-expression of BCL6B dramatically attenuated the inflammation and fibrosis in damaged liver. In contrast, knockdown of BCL6B significantly aggravated the fibrosis with liver injury. In addition, in vitro studies demonstrated that BCL6B inhibited the activation of hepatic stellate cells though upregulation of hepatocyte growth factor (HGF).ConclusionThe expression BCL6B was increased in hepatic fibrogenic tissues. BCL6B plays a pivotal role in preventing liver from inflammation and fibrogenesis, mechanism of which may be enhanced HGF pathway.