Miura Norimasa, Sato Reina, Tsukamoto Tomoe, Shimizu Mika, Kabashima Hiroko, Takeda Miho, Takahashi Shunsaku, Harada Tomomi, West James E, Drabkin Harry, Mejia Jose E, Shiota Goshi, Murawaki Yoshikazu, Virmani Arvind, Gazdar Adi F, Oshimura Mitsuo, Hasegawa Junichi
Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.
BMC Mol Biol. 2009 Feb 2;10:5. doi: 10.1186/1471-2199-10-5.
We attempted to clone candidate genes on 10p 14-15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid.
RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells.
These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.
我们试图通过外显子捕获技术,利用覆盖10p14 - 15区域的3个细菌人工染色体(BAC)克隆来克隆可能调控hTERT表达的候选基因。获得20个外显子后,我们检测了从原代培养的人肝细胞和肝癌细胞系中克隆的RGM249(RGM:miRNAs的RNA基因)的功能。我们证实了由Dicer酶切产生的约20 bp产物,并通过用全长双链RNA、基因特异性设计的小干扰RNA(siRNA)和短发夹RNA(shRNA)表达质粒转染肝癌细胞系,研究了该克隆基因的功能及其与hTERT表达的关系。
RGM249在癌细胞系中呈现出与hTERT相似的以癌症为主的强烈表达,而在无端粒酶活性的人原代肝细胞中表达非常微弱。该基因被预测为非编码前体RNA基因。有趣的是,RGM249双链RNA、siRNA和shRNA抑制了超过80%的hTERT mRNA表达。相反,过表达该基因的原代培养细胞中hTERT mRNA表达没有显著变化;该基因的过表达在低分化细胞中强烈抑制了hTERT mRNA。
这些发现表明,RGM249可能是一个参与hTERT上游分化和功能的微小RNA前体基因。