Yan Z Y, Sun X C
Department of Pathology, Nanyang City Center Hospital, Henan Province, Nanyang 473009, China.
Zhonghua Bing Li Xue Za Zhi. 2018 Apr 8;47(4):284-290. doi: 10.3760/cma.j.issn.0529-5807.2018.04.011.
To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network. Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44(-)CD133(-) and CD44(+) CD133(+) cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation. LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens(<0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis(<0.05), and miR-145 was found related to tumor size and tumor location(<0.05). CD44(+) CD133(+) cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44(+) CD133(+) cells were significantly increased, while miR-145 was decreased compared with CD44(-)CD133(-)cells(<0.05). The levels of CD44, CD133, lnc-ROR in CD44(+) CD133(+) cells were significantly reduced upon cell adherence, while miR-145 was significantly increased(<0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy. Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.
为研究通过结合miR-145发挥ceRNA作用的lincRNA-ROR对下游基因Oct4、Sox2和Nanog表达及结肠癌干细胞相关生物学特性的影响，并阐明该分子调控网络的临床意义。2014年至2016年期间，在河南省南阳市中心医院和南阳第二医院收集了52例结直肠癌组织及癌旁组织。采用实时定量聚合酶链反应（qPCR）检测结直肠癌组织及分离的结肠癌细胞中lincRNA-ROR和miR-145的表达。分析lincRNA-ROR、miR-145表达与结肠癌临床病理特征之间的相关性。通过流式细胞术从SW1116中分离出CD44(-)CD133(-)和CD44(+)CD133(+)细胞。采用qPCR检测细胞中CD44、CD133、Oct4、Sox2、Nanog、lincRNA-ROR和miR-145的表达。通过生物信息学、双荧光素酶报告基因检测、qPCR和蛋白质免疫印迹分析lincRNA-ROR、miR-145、Oct4、Sox2和Nanog之间的关系。采用MTT法、集落形成实验检测沉默lincRNA-ROR对结肠癌干细胞增殖和化疗敏感性的影响。在结肠癌标本中，lincRNA-ROR经常上调，且与miR-145下调呈负相关（<0.05）。lincRNA-ROR与肿瘤大小、淋巴结转移和远处转移相关（<0.05），miR-145与肿瘤大小和肿瘤位置相关（<0.05）。通过流式细胞术成功从SW1116中分离出CD44(+)CD133(+)细胞。与CD44(-)CD133(-)细胞相比，CD44(+)CD133(+)细胞中CD44、CD133、Oct4、Sox2、Nanog、lincRNA-ROR水平显著升高，而miR-145水平降低（<0.05）。细胞贴壁后，CD44(+)CD133(+)细胞中CD44、CD133、lnc-ROR水平显著降低，而miR-145显著升高（<0.05）。生物信息学分析显示，lincRNA-ROR与核心转录因子Oct4、Sox2和Nanog共享miRNA反应元件。miR-145显著抑制lincRNA-ROR、Oct4、Sox2和Nanog的表达。沉默lincRNA-ROR显著抑制结肠癌干细胞增殖并增加其对化疗的敏感性。Linc-ROR作为关键的ceRNA，可防止核心转录因子如Oct4、Sox2、Nanog在结肠癌干细胞中受到miR-145介导的抑制，并调节细胞增殖和化疗敏感性。这些数据可能为深入了解结肠癌发生发展过程中基因网络各成分的病理生理相互作用提供线索，并可能在未来带来新的治疗方法。
