Spatiotemporal modulation of SMAD4 by HBx is required for cellular proliferation in hepatitis B-related liver cancer.

PURPOSE: Hepatitis B virus (HBV) plays a crucial role in the progression of hepatocellular carcinoma (HCC). It is known that HBV-encoded X protein (HBx) can induce genetic alterations in some oncogenes and that SMAD4 is relevant for the development of some cancers, especially HBV-related HCC. Previously, it has been reported that HBx can promote SMAD4 protein expression in liver fibrosis and HCC but, as yet, its regulatory mechanism has not been fully elucidated. Here, we aimed to investigate the correlation between and regulatory mechanism behind HBx and SMAD4 in HCC. METHODS: mRNA and protein expression of SMAD4 in HCC tissues was detected by qRT-PCR, Western blotting and IHC. CCK-8 and colony forming assays, as well as xenograft murine models were used to evaluate the effects of HBx and SMAD4 on the proliferation and tumorigenicity of HCC cells. Luciferase reporter, immunofluorescence, Co-IP and truncation assays were performed to assess the regulatory relationship between HBx and SMAD4. RESULTS: We found that SMAD4 was highly expressed in HBV-positive HCC patient samples and correlated with a poor prognosis. The proliferation of HCC cells with a high SMAD4 expression was found to be enhanced in vitro and in vivo, and knocking down HBx while replenishing SMAD4 rescued HCC cell proliferation. Mechanically, we found that HBx regulates SMAD4 expression at the transcriptional level via TFII-I and can bind to SMAD4 to repress its ubiquitination. The binding region comprised the MH2 domain of SMAD4. Furthermore, we found that SMAD4 can promote HBx expression through a positive feedback mechanism. CONCLUSIONS: From our data we conclude that SMAD4 is modulated spatiotemporally via both transcriptional activation and protein stabilization by HBx in HCC cells. Our data shed light on the molecular mechanism underlying HBx-induced hepatocarcinogenesis.

Detection of circulating tumor cells by recombinant virus / 中华实验和临床病毒学杂志

The detection and molecular characterization of circulating tumor cells(CTCs) is one of the most important tool for liquid biopsy, which has the potential to enable non-invasive diagnostic tests for personalized medicine. Commercial platforms represented by CellSearch, the first FDA approved assay, have been considered to be valid for CTCs detection. However, special equipment and consumptive materials are required in the techniques listed above. Besides, most of them can not differentiate between apoptotic and viable cells, which indicates the portion of active and functional CTCs. Therefore, how to develop novel method for CTCs enrichment with metastatic potential has great significance in clinical routine. Telomerase-specific replication-selective oncolytic viruses expressing green fluorescent protein(GFP), including herpes simplex virus and adenovirus, allow the detection for human CTCs in the peripheral blood. After 24 h of transfection with recombinant virus, the tumor cells stably express GFP, and it could be used for CTCs counting by fluorescent microscopy or flow cytometry. Moreover, downstream analysis would be achieved by combination with PCR or DNA sequencing. Recombinant virus enables early detection of metastatic tumor cells, because the fluorescent signal is amplified only in viable, infected CTCs, by viral replication. This GFP-expressing virus-based method is remarkably sensitive, simple, and feasible, and it offers a new opportunity to detect and characterize CTCs in clinical routine.