Spatiotemporal modulation of SMAD4 by HBx is required for cellular proliferation in hepatitis B-related liver cancer
Spatiotemporal modulation of SMAD4 by HBx is required for cellular proliferation in hepatitis B-related liver cancer
复制标题
HBx 对 SMAD4 的时空调节是乙型肝炎相关肝癌细胞增殖所必需的
DOI：
10.1007/s13402-022-00683-8
复制
发表时间：
2022-06-18
影响因子：
6.6
通讯作者：
Yueguo, Li
中科院分区：
文献类型：
--
作者：
Chaomin, Wang;Wenhao, Niu;Yueguo, Li
目的B型肝炎病毒（HBV）在肝细胞癌（HCC）的发生发展中起重要作用。已知HBV编码的X蛋白（HBx）可诱导某些癌基因的遗传改变，并且SMAD 4与某些癌症的发展相关，特别是HBV相关的HCC。此前，已有报道HBx可以促进SMAD 4蛋白在肝纤维化和HCC中的表达，但其调控机制尚未完全阐明。方法采用qRT-PCR、Western blotting和免疫组化方法检测肝癌组织中SMAD 4 mRNA和蛋白的表达。CCK-8和集落形成测定以及异种移植小鼠模型用于评估HBx和SMAD 4对HCC细胞增殖和致瘤性的影响。采用荧光素酶报告基因、免疫荧光、Co-IP和截短法检测HBx和SMAD 4之间的调控关系。结果SMAD 4在HBV阳性的HCC患者中高表达，且与预后不良相关。发现具有高SMAD 4表达的HCC细胞的增殖在体外和体内都得到增强，并且在补充SMAD 4的同时敲低HBx挽救了HCC细胞的增殖。从机制上讲，我们发现HBx通过TFII-I在转录水平上调节SMAD 4的表达，并可以与SMAD 4结合以抑制其泛素化。结合区包含SMAD 4的MH 2结构域。此外，我们发现SMAD 4可以促进HBx的表达，通过正反馈mechanism.Conclusions从我们的数据，我们得出结论，SMAD 4的调节时空通过转录激活和蛋白稳定的HBx在肝癌细胞。我们的数据揭示了HBx诱导肝癌发生的分子机制。
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.