Toiyama Yuji, Mizoguchi Akira, Kimura Kazushi, Hiro Junichirou, Tutsumi Tomonari, Inoue Yasuhiro, Miki Chikao, Kusunoki Masato
Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
Int J Oncol. 2009 Oct;35(4):709-15. doi: 10.3892/ijo_00000383.
We identified that whirlin is localized to chromosome 9q32-33, and is up-regulated in colorectal cancer tissues by using oligonucleotide array techniques and the Sosui system (http://www.tuat.ac.jp/~mitaku/sosui/). The deduced 920-amino acid protein encoded by the whirlin gene contains three PDZ domains and a proline-rich region that separates PDZ2 from PDZ3, which is located at the C terminus. As previously reported, human whirlin gene is alternatively spliced to form a long and a short transcript in situ hybridization. The sequence of the encoded protein shows that the short C-terminal isoform contains one PDZ domain and the proline-rich domain (whirlin isoform 2), whereas the long isoform is composed of all three PDZ domains and the proline-rich domain (whirlin isoform 1). The gene expression of whirlin was found to be up-regulated in colorectal cancer tissues compared with matched normal colon tissues by semi-quantitative RT-PCR (P<0.05). Western blotting detected whirlin protein with a molecular mass of 49.3 kDa in colorectal cancer samples, suggesting that the whirlin protein overexpressed in colorectal cancer samples is the short C-terminal isoform 2. Its expression was recognized in colorectal cancer cell lines and was increased in accordance with tumor progression in colorectal cancer patients. Immunohistochemistry showed high levels of staining for whirlin isoform 2 only in the mucosal glands in colon cancers, but this protein was barely detected in normal colonic glands. Immunoelectron microscopic findings showed that whirlin isoform 2 is localized on plasma membranes and endoplasmic reticulum membranes, but not in the nuclei. Tissue microarrays showed that whirlin isoform 2 is abundantly expressed in colon cancers with lymph node metastasis compared with those without lymph node metastasis, and overexpression of this protein was associated with tumor progression. In conclusion, we demonstrated that whirlin isoform 2 is highly expressed in colon cancer tissues and that it is related to tumor progression.
我们通过使用寡核苷酸阵列技术和Sosui系统（http://www.tuat.ac.jp/~mitaku/sosui/）确定，whirlin定位于9号染色体q32 - 33，且在结直肠癌组织中上调。whirlin基因编码的推导920个氨基酸的蛋白质包含三个PDZ结构域和一个富含脯氨酸的区域，该区域将位于C端的PDZ2与PDZ3分隔开。如先前报道，人whirlin基因通过原位杂交可选择性剪接形成长转录本和短转录本。编码蛋白的序列表明，短C端异构体包含一个PDZ结构域和富含脯氨酸的结构域（whirlin异构体2），而长异构体由所有三个PDZ结构域和富含脯氨酸的结构域组成（whirlin异构体1）。通过半定量RT-PCR发现，与匹配的正常结肠组织相比，whirlin在结直肠癌组织中的基因表达上调（P<0.05）。蛋白质印迹法在结直肠癌样本中检测到分子量为49.3 kDa的whirlin蛋白，表明在结直肠癌样本中过表达的whirlin蛋白是短C端异构体2。其表达在结直肠癌细胞系中得到证实，并且在结直肠癌患者中随肿瘤进展而增加。免疫组织化学显示，仅在结肠癌的黏膜腺体中whirlin异构体2有高水平染色，但在正常结肠腺体中几乎检测不到该蛋白。免疫电子显微镜结果显示，whirlin异构体2定位于质膜和内质网膜上，而不在细胞核中。组织微阵列显示，与无淋巴结转移的结肠癌相比，whirlin异构体2在有淋巴结转移的结肠癌中大量表达，且该蛋白的过表达与肿瘤进展相关。总之，我们证明了whirlin异构体2在结肠癌组织中高度表达，并且与肿瘤进展相关。
