Department of General Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250012, P.R. China.
Mol Med Rep. 2013 Nov;8(5):1531-6. doi: 10.3892/mmr.2013.1700. Epub 2013 Sep 24.
The zinc-fingers and homeoboxes protein 1 (ZHX1) consists of 873 amino acid residues, is localized in the cell nucleus and appears to act as a transcriptional repressor. Previous studies have shown that ZHX1 interacts with nuclear factor Y subunit α (NF-YA), DNA methyltransferases (DNMT) 3B and ZHX2, all of which are involved in tumorigenesis. However, the exact role of ZHX1 in tumorigenesis remains unknown. The aim of the current study was to construct a recombinant eukaryotic expression plasmid containing the human ZHX1 (hZHX1) gene and to investigate the biological activities of ZHX1 in hepatocellular carcinoma (HCC). Reverse transcription-polymerase chain reaction (RT‑PCR) was used to amplify the N- and C-terminal fragments (ZHX1‑N and ZHX1‑C, respectively) of the hZHX1 gene. The two PCR fragments were cloned into the pEASY-T1 vector and subcloned into the pcDNA3 plasmid to generate a recombinant pcDNA3‑ZHX1 plasmid. Following identification by enzyme digestion and DNA sequencing, the recombinant pcDNA3‑ZHX1 plasmid was transfected into SMMC-7721 cells. The level of ZHX1 expression was detected by RT-PCR and western blot analysis. Cell growth curve assays were used to evaluate the effect of ZHX1 on cell proliferation. Moreover, the differential expression of ZHX1 between cancer and adjacent cirrhotic liver tissue was investigated by quantitative PCR (qPCR). Enzyme digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid, pcDNA3‑ZHX1. qPCR and western blot analysis demonstrated that ZHX1 was efficiently expressed in SMMC-7721 cells and overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells. In addition, reduced ZHX1 expression is widespread among cancer tissues from HCC patients. In conclusion, a recombinant eukaryotic expression plasmid, pcDNA3‑ZHX1, was successfully constructed. In addition, the current results indicate that a low expression of ZHX1 may be responsible for hepatocarcinogenesis.
锌指和同源盒蛋白 1（ZHX1）由 873 个氨基酸残基组成，定位于细胞核内，似乎作为转录抑制剂发挥作用。先前的研究表明，ZHX1 与核因子 Y 亚基 α（NF-YA）、DNA 甲基转移酶（DNMT）3B 和 ZHX2 相互作用，所有这些都与肿瘤发生有关。然而，ZHX1 在肿瘤发生中的确切作用仍不清楚。本研究旨在构建含有人 ZHX1（hZHX1）基因的重组真核表达质粒，并研究 ZHX1 在肝细胞癌（HCC）中的生物学活性。逆转录-聚合酶链反应（RT-PCR）用于扩增 hZHX1 基因的 N 端和 C 端片段（ZHX1-N 和 ZHX1-C）。将两个 PCR 片段克隆到 pEASY-T1 载体中，并亚克隆到 pcDNA3 质粒中，以生成重组 pcDNA3-ZHX1 质粒。经酶切和 DNA 测序鉴定后，将重组 pcDNA3-ZHX1 质粒转染至 SMMC-7721 细胞。通过 RT-PCR 和 Western blot 分析检测 ZHX1 的表达水平。细胞生长曲线分析用于评估 ZHX1 对细胞增殖的影响。此外，通过定量 PCR（qPCR）研究 ZHX1 在癌症和相邻肝硬化组织之间的差异表达。酶切和 DNA 测序证实重组质粒 pcDNA3-ZHX1 的成功构建。qPCR 和 Western blot 分析表明，ZHX1 在 SMMC-7721 细胞中高效表达，过表达 ZHX1 可能抑制 SMMC-7721 细胞的增殖。此外，肝癌患者的癌症组织中 ZHX1 表达降低较为普遍。综上所述，成功构建了重组真核表达质粒 pcDNA3-ZHX1。此外，目前的结果表明，ZHX1 表达水平降低可能与肝癌的发生有关。
