Calcium ions affect the hepatitis B virus core assembly

Previous report showed that cytosolic Ca2+ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca2+ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.     

HBx和mIL-12双基因重组腺病毒的构建

目的 构建携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12.方法 采用酶切、连接的方法分别将基因HBx和mIL-12连接到穿梭质粒载体pAdenoVator-CMV5.将构建正确的穿梭质粒pAdV-HBx-mIL-12经EcoR Ⅰ酶切线性化后,转化于含腺病毒骨架质粒pAdenoVatorΔE1/E3的BJ5183感受态细菌,实现细菌内同源重组.将鉴定正确的重组腺病毒质粒pAd-HBx-mIL-12经Pac Ⅰ酶切线性化后,以脂质体法转染至293细胞,包装并扩增获得携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12.结果 经鉴定携带HBx和mIL-12双基因的重组腺病毒构建成功,并可在293细胞中有效表达.结论 成功构建了携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12,为探讨其联合抗肿瘤机制及后续基因治疗奠定了基础.     

Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas

Hepatocellular carcinoma (HCC) is the fourth most common form of cancer in the Korean population, caused primarily by infection with either the Hepatitis B or C virus. Progression of this disease is frequently associated with mutations in either phosphoinositide‐3‐kinase, catalytic, alpha (PIK3CA) or hepatitis B virus X (HBx) gene. Previous studies have examined the frequency of PIK3CA mutations in HCC, although the clinical significance of these mutations has not been studied in a Korean population. In addition, HBx appears to play a key role in modulating a wide range of cellular functions, leading to HCC. In this study, we examined microdissected tumor samples from 50 HCC patients who underwent hepatectomy at Keimyung University Dongsan Medical Center. These patients were screened for mutations in PIK3CA and HBx to identify the clinical outcomes associated with these mutations. Exons 9 and 20 of PIK3CA and the entirety of HBx were screened for mutations by polymerase chain reaction and direct DNA sequencing. PIK3CA mutations were detected in 7 of 50 patients (14%). Among the 42 patients who were seropositive for hepatitis B, 17 (40.5%) had HBx mutations and 4 (9.52%) had mutations in PIK3CA. PIK3CA mutations were strongly correlated with tumor size. Patients harboring HBx mutations exhibited a longer time to recurrence; this difference was statistically significant not only in comparison with the PIK3CA mutation but also compared with those without any mutations. This result suggests a role for PIK3CA and HBx mutations as prognostic markers in HCC.     

HBx及其截短体在乙型肝炎病毒复制中的作用

HBV感染作为引起慢性乙型肝炎、乙肝后肝硬化、原发性肝癌(hepatocellular carcinoma,HCC)的起始因素,已成为世界性的健康问题.据统计,目前世界上已有超过5亿人感染HBV,每年有1百万人死于乙肝相关疾病.HBx蛋白作为HBV的一个多功能调节蛋白,已被证实在HCC的发生过程中起了重要作用.近年来,关于乙肝病毒X蛋白(HBV X protein,HBx)影响HBV的复制研究也有了一定的进展.同时,越来越多的HBx截短体在肝病发展过程中的作用也被重视.本文将对HBx及其截短体在HBV复制中的作用作一综述.     

稳定表达HBx的人肝细胞系7702的建立

目的 构建HBx表达载体,筛选可稳定表达HBx的人肝细胞(HL)-7702细胞系。 方法 采用RT-PCR法扩增HBx 基因片段,并将其连接至pIRES载体,经酶切和测序鉴定pIRES-HBx重组质粒序列。然后,分别采用Real-time PCR和Western blot技术检测验证重组质粒的正确性。最后,应用G418抗生素筛选稳定表达HBx的HL-7702细胞系。结果 经PCR扩增得到正确的HBx片段,成功构建pIRES-HBx质粒,将重组质粒转染HEK 293 细胞和HL-7702细胞均实现了HBx过量表达;经免疫荧光法鉴定,我们获得了稳定大量表达HBx的HL-7702细胞系。结论 成功构建的pIRES-HBx表达载体能在HL-7702细胞稳定表达HBx,为后续研究提供了基础实验工具。     

Oncogenic role of p21 in hepatocarcinogenesis suggests a new treatment strategy

A well-known tumor suppressor, p21, acts parado-xically by promoting tumor growth in some cellular conditions. These conflicting functions have been demonstrated in association with the HBx gene and in hepatocarcinogenesis. The molecular behavior of p21 depends on its subcellular localization. Nuclear p21 may inhibit cell proliferation and be proapoptotic, while cytoplasmic p21 may have oncogenic and anti-apoptotic functions. Because most typical tumor suppressive proteins also have different effects according to subcellular localization, elucidating the regulatory mechanisms underlying nucleo-cytoplasmic transport of these proteins would be significant and may lead to a new strategy for anti-hepatocellular carcinoma(HCC) therapy. Chromosome region maintenance 1(CRM1) is a major nuclear export receptor involved in transport of tumor suppressors from nucleus to cytoplasm. Expression of CRM1 is enhanced in a variety of malignancies and in vitro studies have shown the efficacy of specific inhibition of CRM1 against cancer cell lines. Interestingly, interferon may keep p21 in the nucleus; this is one of the mechanisms of its anti-hepatocarcinogenic function. Here we review the oncogenic property of p21, which depends on its subcellular localization, and discuss the rationale underlying a new strategy for HCC treatment and prevention.     

乙肝病毒X蛋白突变对其反式激活作用的影响

原发性肝癌（hepatocellular carcinoma，HCC）是世界上最常见的肿瘤之一，慢性乙肝病毒（HBV）感染是HCC发生发展的一个主要危险因素。乙型肝炎病毒X蛋白（HBx）与HCC的形成密切相关，其特有的反式激活功能被认为足HBV与HCC关联的主要因素。HBV相关的肝癌患者组织和血清中普遍存在HBx蛋白的自然突变，HBx蛋白的突变改变了全长HBx蛋白的反式激活活性，使其在调控细胞生长和增殖方面产生了不同的影响。