Fujino Yasuteru, Takeishi Shunsaku, Nishida Kensei, Okamoto Koichi, Muguruma Naoki, Kimura Tetsuo, Kitamura Shinji, Miyamoto Hiroshi, Fujimoto Akiko, Higashijima Jun, Shimada Mitsuo, Rokutan Kazuhito, Takayama Tetsuji
Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Department of Pathophysiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Cancer Sci. 2017 Mar;108(3):390-397. doi: 10.1111/cas.13152.
A majority of early colorectal cancers (CRCs) with submucosal invasion undergo surgical operation, despite a very low incidence of lymph node metastasis. Our study aimed to identify microRNAs (miRNAs) specifically responsible for lymph node metastasis in submucosal CRCs. MicroRNA microarray analysis revealed that miR-100 and miR-125b expression levels were significantly lower in CRC tissues with lymph node metastases than in those without metastases. These results were validated by quantitative real-time PCR in a larger set of clinical samples. The transfection of a miR-100 or miR-125b inhibitor into colon cancer HCT116 cells significantly increased cell invasion, migration, and MMP activity. Conversely, overexpression of miR-100 or miR-125b mimics significantly attenuated all these activities but did not affect cell growth. To identify target mRNAs, we undertook a gene expression array analysis of miR-100-silenced HCT116 cells as well as negative control cells. The Ingenuity Pathway Analysis, TargetScan software analyses, and subsequent verification of mRNA expression by real-time PCR identified mammalian target of rapamycin (mTOR) and insulin-like growth factor 1 receptor (IGF1R) as direct, and Fas and X-linked inhibitor-of-apoptosis protein (XIAP) as indirect candidate targets for miR-100 involved in lymph node metastasis. Knockdown of each gene by siRNA significantly reduced the invasiveness of HCT116 cells. These data clearly show that downregulation of miR-100 and miR-125b is closely associated with lymph node metastasis in submucosal CRC through enhancement of invasion, motility, and MMP activity. In particular, miR-100 may promote metastasis by upregulating mTOR, IGF1R, Fas, and XIAP as targets. Thus, miR-100 and miR-125b may be novel biomarkers for lymph node metastasis of early CRCs with submucosal invasion.
尽管黏膜下浸润的早期结直肠癌（CRC）发生淋巴结转移的几率很低，但大多数此类癌症患者仍接受了外科手术。我们的研究旨在确定在黏膜下CRC中对淋巴结转移有特殊作用的微小RNA（miRNA）。微小RNA微阵列分析显示，发生淋巴结转移的CRC组织中miR-100和miR-125b的表达水平显著低于未发生转移的组织。这些结果在更多临床样本中通过定量实时PCR得到了验证。将miR-100或miR-125b抑制剂转染至结肠癌HCT116细胞中，可显著提高细胞的侵袭、迁移及基质金属蛋白酶（MMP）活性。相反，miR-100或miR-125b模拟物的过表达可显著减弱所有这些活性，但不影响细胞生长。为了鉴定靶mRNA，我们对miR-100沉默的HCT116细胞以及阴性对照细胞进行了基因表达阵列分析。通过Ingenuity Pathway Analysis、TargetScan软件分析以及随后通过实时PCR对mRNA表达进行验证，确定雷帕霉素哺乳动物靶蛋白（mTOR）和胰岛素样生长因子1受体（IGF1R）为miR-100在淋巴结转移中直接作用的候选靶标，而Fas和X连锁凋亡抑制蛋白（XIAP）为间接候选靶标。通过小干扰RNA（siRNA）敲低每个基因可显著降低HCT116细胞的侵袭性。这些数据清楚地表明，miR-100和miR-125b的下调通过增强侵袭、运动及MMP活性与黏膜下CRC的淋巴结转移密切相关。特别是，miR-100可能通过上调作为靶标的mTOR、IGF1R、Fas和XIAP来促进转移。因此，miR-100和miR-125b可能是黏膜下浸润早期CRC淋巴结转移的新型生物标志物。
