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.

Chlamydia muridarum respiratory infection induces macrophage infiltration and polarization toward M1 phenotype / 中华微生物学和免疫学杂志

Objective:To investigate the infiltration and polarization of macrophages in mice during Chlamydia muridarum ( Cm) respiratory infection. Methods:C57BL/6 mice were intranasally infected with 1×10 3 inclusion-forming units (IFU) of Cm to establish the mouse model of Cm respiratory tract infection. The percentages of CD45 + F4/80 + cells and the macrophages expressing CD86, major histocompatibility complex Ⅱ (MHC), inducible nitric oxide synthase (iNOS) and CD206 were detected by flow cytometry. Expression of iNOS, CD206 and CCL2 at mRNA level was detected by real-time quantitative PCR. Results:Cm respiratory tract infection induced the increase of macrophages in mouse lung tissues. Compared with uninfected group, CD45 + F4/80 + macrophages were increased significantly from day 3 and reached the peak on day 7 after Cm infection. Moreover, the expression of CD86, MHCⅡ and CCL2 was increased, and the macrophages were polarized to M1 phenotype. However, the expression of M2 macrophage marker CD206 was decreased gradually. Further studies showed that iNOS expression, the indicator of M1 macrophage activation, was increased after Cm infection and reached to the top on day 7. Conclusions:Cm respiratory infection could induce the infiltration of macrophages in lung tissues and promote the polarization of macrophages to M1 phenotype.