Chen Xiang-Yu, Li Jian-sheng, Ma Jun, Duan Fang-ling, Zhong Peng
Department of Digestive Disease, Institute of Clinical Medical Research of Henan Province, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Chin Med J (Engl). 2006 Oct 20;119(20):1709-14.
We have used suppression subtractive hybridization to construct a subtracted cDNA library of hepatocellular carcinoma (HCC) and isolated a panel of differential expression sequence tag (ESTs). By using bioinformatics and rapid amplification of cDNA ends (RACE), we found a novel HCC-associated gene IDD01. To further investigate its function, a recombinant eukaryotic vector pEGFP/ORF was constructed and transfected into the HepG2 cell line.
The open reading frame (ORF) of IDD01 was amplified by RT-PCR, digested with Bamh I and Hind III, and subcloned into the pEGFP-C1 vector. The ligation reaction was conducted with T4 DNA ligase, and the recombinant vector was named pEGFP/ORF. Untransfer control (control group), pEGFP-C1 (HepG2/C1 group) and pEGFP/ORF (HepG2/ORF group) transfer groups were designed. Gene transfer was conducted with lipofectamine. To obtain stable transfection in HepG2 cells, selection was initiated with 500 microg/ml G418. Cellular IDD01 mRNA levels were assayed by semi-quantitative RT-PCR. The MTT colorimetric method and flow cytometry were used to determine the cell proliferation. The tumorigenic potential of transformed cells was determined from their ability to grow as anchorage-independent colonies on soft agar. Transient transfections were performed to observe subcellular location of GFP-IDD01 fusion protein.
A 778 bp specific band of ORF was obtained by RT-PCR, and the positive clone of recombinant plasmid pEGFP/ORF (5.5 Kb) was identified by restriction endonuclease cleavage and sequence. The brightness ratio of IDD01 mRNA was not obvious between control and pEGFP/C1 groups, whereas the ratio of pEGFP/ORF was higher than that in the other two groups. After culture for 24 - 72 hours, the A(490) values in pEGFP/ORF were higher than those in the other two groups (P < 0.01). On histograms of flow cytometry, the S phase ratio of HepG2/ORF cells was significantly higher than that of the control and HepG2/C1 groups. The HepG2/ORF cells were able to form more colonies in soft agar compared with other HepG2 cell lines (P < 0.01). GFP-IDD01 fusion protein predominantly localized in the plasma, whereas EGFP protein diffused all over the cell.
The IDD01 gene is a positive effector in cell proliferation and contributes to the carcinogenesis and progression of HCC. This gene may serve as a potential target for pharmaceutical intervention of HCC.
我们运用抑制性消减杂交技术构建了肝细胞癌（HCC）的消减cDNA文库，并分离出一组差异表达序列标签（ESTs）。通过生物信息学和cDNA末端快速扩增（RACE）技术，我们发现了一个新的HCC相关基因IDD01。为进一步研究其功能，构建了重组真核载体pEGFP/ORF并转染至HepG2细胞系。
通过RT-PCR扩增IDD01的开放阅读框（ORF），用Bamh I和Hind III进行酶切，亚克隆至pEGFP-C1载体。用T4 DNA连接酶进行连接反应，将重组载体命名为pEGFP/ORF。设计未转染对照（对照组）、pEGFP-C1（HepG2/C1组）和pEGFP/ORF（HepG2/ORF组）转染组。用脂质体进行基因转染。为在HepG2细胞中获得稳定转染，用500μg/ml G418进行筛选。通过半定量RT-PCR检测细胞IDD01 mRNA水平。采用MTT比色法和流式细胞术测定细胞增殖情况。根据转化细胞在软琼脂上形成非锚定依赖性集落的能力来确定其致瘤潜力。进行瞬时转染以观察GFP-IDD01融合蛋白的亚细胞定位。
通过RT-PCR获得了一条778 bp的ORF特异性条带，经限制性内切酶酶切和测序鉴定出重组质粒pEGFP/ORF（5.5 Kb）的阳性克隆。对照组和pEGFP/C1组之间IDD01 mRNA的亮度比值不明显，而pEGFP/ORF组的比值高于其他两组。培养24 - 72小时后，pEGFP/ORF组的A(490)值高于其他两组（P < 0.01）。在流式细胞术直方图上，HepG2/ORF细胞的S期比例显著高于对照组和HepG2/C1组。与其他HepG2细胞系相比，HepG2/ORF细胞在软琼脂中能够形成更多集落（P < 0.01）。GFP-IDD01融合蛋白主要定位于细胞质膜，而EGFP蛋白则在细胞内弥漫分布。
IDD01基因是细胞增殖的正性效应因子，有助于HCC的发生和发展。该基因可能成为HCC药物干预的潜在靶点。
