Yu Bo, Ye Xuan, Du Qiong, Zhu Bin, Zhai Qing, Li Xin-Xiang
Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.
Department of Pharmacy, Fudan University Shanghai Cancer Center, Shanghai, China.
Cell Physiol Biochem. 2017;41(6):2489-2502. doi: 10.1159/000475941. Epub 2017 May 4.
BACKGROUND/AIMS: The long non-coding RNA colorectal neoplasia differentially expressed (CRNDE) contributes to the proliferation and migration of tumors. However, its molecular mechanism underlying gastric cancer remains unknown. In the present study, we investigated whether CRNDE was involved in the development of colorectal cancer via the binding of microRNA (miR)-217 with transcription factor 7-like 2 (TCF7L2) to enhance the Wnt signaling pathway.
Quantitative polymerase chain reaction was used to detect CRNDE, miR-217 and TCF7L2 in colorectal cancer tissues and cells. The CCK-8 assay, wound healing assay, and Transwell assay were used to detect cell proliferation, migration and invasion, respectively. Western blotting and luciferase activity assays were used to identify CRNDE and TCF7L2 as one of the direct targets of miR-217. The activity of the Wnt/β-catenin signaling pathway was analyzed by the TOPflash assay, and the subcellular localization of β-catenin and TCF7L2 was analyzed by western blotting and confocal microscopy.
In this study, we found that high expression of CRNDE is negatively correlated with low expression of miR-217 in colorectal cancer tissue and colorectal cancer cells. The dual luciferase reporter analysis showed that miR-217 is bound to CRNDE and TCF7L2 and negatively regulate their expression. CRNDE down-regulation inhibited the cell proliferation, migration and invasion in vitro and in vivo and the inhibitions were both completely blocked after miR-217 inhibition or TCF7L2 overexpression. Finally, TOPflash analysis showed that the activity of Wnt/β-catenin signaling is inhibited by CRNDE down-regulation and rescued by TCF7L2 over-expression. Consistently immunostaining and western blotting analysis showed that the expression of b-catenin and TCF7L2 in the nucleus was significantly decreased by CRNDE down-regulation and was rescued by TCF7L2 over-expression.
The present study suggest that CRNDE involves in the cell proliferation, migration and invasion of colorectal cancer cells via increasing the expression of TCF7L2 and activity of Wnt/β-catenin signaling through binding miR-217 competitively.
背景/目的：长链非编码RNA结直肠肿瘤差异表达基因（CRNDE）促进肿瘤的增殖和迁移。然而，其在胃癌发生发展中的分子机制尚不清楚。在本研究中，我们探究了CRNDE是否通过与微小RNA（miR）-217结合并与转录因子7样2（TCF7L2）相互作用增强Wnt信号通路，从而参与结直肠癌的发生发展。
采用定量聚合酶链反应检测结直肠癌组织及细胞中CRNDE、miR-217和TCF7L2的表达水平。分别采用CCK-8法、伤口愈合实验和Transwell实验检测细胞的增殖、迁移和侵袭能力。通过蛋白质免疫印迹法和荧光素酶活性检测实验鉴定CRNDE和TCF7L2是miR-217的直接靶标之一。采用TOPflash实验分析Wnt/β-连环蛋白信号通路的活性，通过蛋白质免疫印迹法和共聚焦显微镜观察分析β-连环蛋白和TCF7L2的亚细胞定位。
在本研究中，我们发现结直肠癌组织及细胞中CRNDE的高表达与miR-217的低表达呈负相关。双荧光素酶报告基因分析表明，miR-217与CRNDE和TCF7L2结合并负向调节它们的表达。下调CRNDE可抑制体外及体内细胞的增殖、迁移和侵袭，而抑制miR-217或过表达TCF7L2均可完全阻断这种抑制作用。最后，TOPflash分析表明，下调CRNDE可抑制Wnt/β-连环蛋白信号通路的活性，而过表达TCF7L2可使其恢复。一致地，免疫染色和蛋白质免疫印迹分析表明，下调CRNDE可显著降低细胞核中β-连环蛋白和TCF7L2的表达，而过表达TCF7L2可使其恢复。
本研究表明，CRNDE通过竞争性结合miR-217，增加TCF7L2的表达及Wnt/β-连环蛋白信号通路的活性，从而参与结直肠癌细胞的增殖、迁移和侵袭。
