miR-144 通过靶向 Kruppel 样因子 4 抑制结肠癌干细胞干性的初步研究。
A Preliminary Study of miR-144 Inhibiting the Stemness of Colon Cancer Stem Cells by Targeting Krüppel-Like Factor 4.
出版信息
J Biomed Nanotechnol. 2020 Jul 1;16(7):1102-1109. doi: 10.1166/jbn.2020.2952.
Colon cancer is a prevalent clinical malignant tumor of the digestive system. The current study aims to explore the miR-144 expression in colorectal cancer (CRC) cell lines and CRC stem cells (CSCs) and to explore its effect on the stemness of CSCs and the targeted regulation of Krüppel-like factor 4 (KLF4). Use qRT-PCR to detect the expression level of miR-144 in CRC cells SW480, HCT116, and H129 and the healthy colon cell NCM460. The CSCs that were used were cultured in HCT116 cells. Use western blot to explore the expressions of Nanog, SOX2, and OCT4 stemness marker protein. After it was transfected with miR-144 mimics or KLF4 plasmid, use MTT to explore the cell viability of CSCs, use flow cytometry to evaluate apoptosis, and use transwell assay to evaluate the ability of invasive of CSCs. The targeting effect of miR-144 on the KLF4 gene was verified using TargetScan prediction and the double-luciferase reporter gene test. Use qRT-PCR to evaluate the role of miR-144 mimics on KLF4 mRNA expression in CSCs. The qRT-PCR results exhibited that the miR-144 expression in CRC cells was higher than that in the healthy colon cell line. The expressions of OCT4, Nanog, and SOX2 stem cell markers were up-regulated in CSCs, and the expression of miR144 increased in CSCs. The cell viability, apoptosis, and invasion of CSCs increased after miR-144 was transfected. The TargetScan prediction and double-luciferase reporter gene assay confirmed that miR-144 was targeted by KLF4, and the expression of KLF4 mRNA in the miR-144 mimics group reduced. Moreover, the overexpression of KLF4 could partially reverse the role of miR-144 mimics on CSCs. In summary, miR-144 was highly expressed in CRC cell lines and CSCs, and the overexpression of miR-144 in CSCs significantly promoted the proliferation of CSCs, inhibited its apoptosis, and promoted its invasion ability. In addition, its preliminary mechanism, possibly through negative regulation KLF4, promotes the stemness of CSCs, and miR-144 is likely to be a potential target for eliminating CSC from CRC treatment.
结肠癌是消化系统常见的恶性肿瘤之一。本研究旨在探讨 miR-144 在结直肠癌细胞系和结直肠肿瘤干细胞（CSC）中的表达，及其对 CSC 干性的影响和对 Krüppel 样因子 4（KLF4）的靶向调控。用 qRT-PCR 检测 miR-144 在 CRC 细胞株 SW480、HCT116 和 H129 及正常结肠细胞 NCM460 中的表达水平。CSC 培养于 HCT116 细胞中。用 Western blot 检测干性标志物蛋白 Nanog、SOX2 和 OCT4 的表达。转染 miR-144 模拟物或 KLF4 质粒后，用 MTT 法检测 CSC 的细胞活力，用流式细胞术评估细胞凋亡，用 Transwell 小室实验评估 CSC 的侵袭能力。用 TargetScan 预测和双荧光素酶报告基因试验验证 miR-144 对 KLF4 基因的靶向作用。用 qRT-PCR 评价 miR-144 模拟物对 CSC 中 KLF4mRNA 表达的作用。qRT-PCR 结果显示，CRC 细胞中的 miR-144 表达高于正常结肠细胞系。CSC 中 OCT4、Nanog 和 SOX2 干细胞标志物表达上调，miR144 在 CSC 中表达增加。转染 miR-144 后，CSC 的细胞活力、凋亡和侵袭均增加。TargetScan 预测和双荧光素酶报告基因试验证实 KLF4 是 miR-144 的靶基因，miR-144 模拟物组中 KLF4mRNA 的表达降低。此外，KLF4 的过表达可部分逆转 miR-144 模拟物对 CSC 的作用。综上所述，miR-144 在 CRC 细胞系和 CSC 中高表达，CSC 中 miR-144 的过表达显著促进 CSC 的增殖，抑制其凋亡，并促进其侵袭能力。此外，其初步机制可能通过负调控 KLF4 促进 CSC 的干性，miR-144 可能成为从 CRC 治疗中消除 CSC 的潜在靶点。
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