Lun Yong-Zhi, Cheng Jun, Chi Qing, Wang Xue-Lei, Gao Meng, Sun Li-DA
Liaoning Provincial University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China.
Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, P.R. China.
Oncol Lett. 2014 Aug;8(2):803-808. doi: 10.3892/ol.2014.2190. Epub 2014 May 28.
C-terminally truncated hepatitis B virus (HBV) middle size surface proteins (MHBs) has been shown to be a transcriptional activator and may be relevant to hepatocarcinogenesis by transactivating gene expression. In the present study, a pcDNA3.1(-)-MHBs vector coding for MHBs truncated at amino acid 167 (MHBs) was constructed and transfected into the HepG2 hepatoma cell line. mRNA and protein expression of MHBs in the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. A cDNA library of genes transactivated by the truncated protein in HepG2 cells was made in pGEM-T Easy using suppression subtractive hybridization. The cDNAs were sequenced and analyzed with BLAST searching against the sequences in GenBank. The results showed that certain sequences, such as that of human proto-oncogene c-Myc, may be involved in tumor development. An expression vector pCAT3/c-Myc containing the chloramphenicol acetyltransferase (CAT) gene under the control of a c-Myc promoter was generated, and the transcriptional transactivating effect of MHBs on the c-Myc promoter was investigated by RT-PCR and western blotting. MHBs was found to upregulate the transcriptional activity of the promoter, as well as transcription and translation of c-Myc. MHBs was also shown to transactivate SV40 immediate early promoter, and transcriptionally transactivate the expression of human c-Myc. These findings provide new directions for studying the biological functions of MHBs, and for a better understanding of the tumor development mechanisms of HBV infection.
C 末端截短的乙型肝炎病毒（HBV）中分子表面蛋白（MHBs）已被证明是一种转录激活因子，可能通过反式激活基因表达与肝癌发生相关。在本研究中，构建了编码在氨基酸 167 处截短的 MHBs（MHBsΔ167）的 pcDNA3.1(-)-MHBs 载体，并将其转染到 HepG2 肝癌细胞系中。通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测细胞中 MHBsΔ167 的 mRNA 和蛋白质表达。利用抑制性消减杂交技术，在 pGEM-T Easy 载体中构建了 HepG2 细胞中被截短蛋白反式激活的基因的 cDNA 文库。对这些 cDNA 进行测序，并通过 BLAST 在 GenBank 中搜索序列进行分析。结果表明，某些序列，如人类原癌基因 c-Myc 的序列，可能参与肿瘤发展。构建了一个在 c-Myc 启动子控制下含有氯霉素乙酰转移酶（CAT）基因的表达载体 pCAT3/c-Myc，并通过 RT-PCR 和蛋白质免疫印迹研究了 MHBsΔ167 对 c-Myc 启动子的转录反式激活作用。发现 MHBsΔ167 上调启动子的转录活性以及 c-Myc 的转录和翻译。还表明 MHBsΔ167 反式激活 SV40 立即早期启动子，并转录反式激活人类 c-Myc 的表达。这些发现为研究 MHBs 的生物学功能以及更好地理解 HBV 感染的肿瘤发展机制提供了新的方向。
