【作者】 张经余； 戴建武； 吕有勇；

【Author】 Zhang Jing-Yu Dai Jian-Wu Lu You-Yong Molecular Oncology Laboratory, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, 100036 Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100080

【机构】 北京市肿瘤防治研究所肿瘤分子生物学实验室北京大学临床肿瘤学院； 中国科学院遗传与发育生物学研究所发育生物学实验室；

【摘要】 目的:Nanog是与小鼠和人胚胎干细胞自我更新和多能性维持相关的转录因子。人Nanog基因有11个假基因包括10个逆转录型假基因和1个复制型假基因。在正常细胞、肿瘤细胞、正常组织、肿瘤组织中对Nanog及其假基因转录情况进行了检测,发现Nanog只在具有胚胎干细胞特性的畸胎瘤PA-1和NTERA-2细胞中转录,而在正常细胞／组织和肿瘤细胞／组织中不转录,但是在肿瘤细胞和肿瘤组织中有 Nanog的假基因8(NANOGP8)转录。NANOGP8具有完整的开放读码框(ORF),理论上预测有蛋白表达,应该是一个逆转录基因,本研究的目的是明确NANOGP8是否为逆转录基因以及在胃癌发生发展中的作用。方法:从癌组织中通过 RT—PCR并结合DNA测序等方法克隆NANOGP8的ORF 全长。测序正确的NANOGP8序列构建原核和真核表达载体, 转染NIH3T3细胞,通过MTT实验检测细胞增殖。进一步用免疫组化方法检测NANOGP8在胃癌组织中的表达变化与肿瘤临床病理分期和预后的关系。结果:构建NANOGP8原核表达载体并获得表达蛋白,通过His纯化,经Western blot 检测证明纯化出来的蛋白是NANOGP8,这表明NANOGP8 可以在体外进行表达。将融合表达载体p Q C X I N～ NANOGP8-GFP转染到NIH3T3细胞中,结果发现融合蛋白定位于细胞核,这一方面说明了NANOGP8是核蛋白,同时也说明NANOGP8在真核细胞里可以表达。利用免疫组化,在人肿瘤细胞系HepG2、OS732中检测到有NANOGP8蛋白表达。进一步通过转染NIH3T3,发现NANOGP8具有促进 NIH3T3细胞增殖的能力,这一作用与Nanog基因相似。在此基础上,用抗NANOGP8抗体进行胃癌组织芯片(tissue array)的免疫组化分析,结果表明NANOGP8在胃癌组织中表达阳性率为70％(56／80),在正常组织中阳性率为5％(7／ 140),这一结果提示NANOGP8异常表达可能与胃癌的发生发展有关。结论:NANOGP8是一个逆转录基因,在肿瘤细胞和胃癌组织中异常表达,可能在肿瘤发生中起重要作用。更多还原

【Abstract】 Objective: Nanog is a transcription factor that plays key roles in the self-renewal and maintenance of pluripotency in mouse and human embryonic stem (ES) cells. Human Nanog pseudogenes comprised 10 processed pseudogenes and one tandem duplicate. Nanog was expressed in PA-1 and NTERA-2 and NANOGP8 was expressed in several cancer cell lines and cancer tissues tested, while no Nanog expression was detected in normal cell lines/ tissues and cancer cell lines/tissues. NANOGP8 may be a retrogene rather than a pseudogene because it has a complete open reading frame. Our objective is to explore whether NANOGP8 is a retrogene and to find the correlation between its expression in gastric cancer and tumor occurrence and development. Methods: RT-PCR and DNA sequencing were used to clone the ORF of NANOGP8 and cell transfection, MTT were used to investigate its functions. Results: We were able to detect its protein expression using anti-Nanog antibody in recombinant Es-cherichia coli. We constructed a NANOGP8 and GFP fu- sion protein and found the fusion protein was localized in the nuclei of transfected NIH3T3 while GFP in the control group was present diffused in the cytoplasm, which indicated that NANOGP8 is a nuclear protein. At the same time, we detected the NANOGP8 in OS732 and HepG2 cell lines. When NANOGP8 was stably transfected into NIH3T3 cells, the cells were promoted to enter into the S stage and, at the same tune, MTT growth assay showed increased cell proliferation. IHC results showed NANOGP8 was over-expressed in 70% (56/80 cases) gastric tumor tissues and 5% (7/140) in matched normal tissues, which indicated NANOGP8 may play important roles in gastric cancer occurrence and development Conclusion: NANOGP8 is a retrogene over-expressed in cancer cell lines and gastric cancer and may play important roles in tumorigenesis.更多还原