ABSTRACT

Emerging reports have shown that microRNAs (miRNAs) function as vital regulators in tumor development via modulating gene expression at the posttranscriptional level. Here, we explored the role and underlying mechanism of miR-663a in the proliferation, migration, invasion, and cancer stem cell-like (CSC) properties of glioma cells. Quantitative reverse transcription PCR (qRT‐PCR) was implemented to detect miR-663a expression in glioblastoma tissues and the adjacent normal tissues. Additionally, gain- and loss-of-function assays of miR-633a were performed on U-251 MG cells or human primary glioblastoma cancer cells (pGBMC1). Cell proliferation, migration, invasion, CSC properties, and profiles of stem cell markers (including CD133, CD44) were examined by the MTT assay, Transwell assay, tumorsphere experiment, and Western blotting, respectively. The dual-luciferase reporter gene assay was performed to testify the targeted relationship between miR-663a and lysine demethylase 2A (KDM2A). The results showed that miR-663a was down-regulated in glioblastoma tissues and cells. Overexpressing miR-663a repressed the proliferation, migration, invasion, CSC properties of U-251 MG cells and pGBMC1, while miR-663a knockdown had the opposite effects. The in-vivo experiment confirmed that miR-663a repressed the growth of U-251 MG cells in nude mice. When cocultured with THP1 cells, U-251 MG cells gained enhanced proliferation, migration, invasion, and CSC properties. MiR-633a overexpression reversed THP1-mediated effects on U-251 MG cells, and reduced the “M2” polarization of THP1 cells. What’s more, Mechanistically, KDM2A was targeted by miR-663a. KDM2A knockdown suppressed the progression and CSC properties of U-251 MG cells in vitro, and dampened TGF-β. Overall, those data revealed that up-regulating miR-663a reduced glioma progression by inhibiting the KDM2A-mediated TGF-β/Smad pathway.

摘要

新出现的报告表明，microRNA (miRNA) 通过在转录后水平调节基因表达，在肿瘤发展中发挥重要调节作用。在这里，我们探讨了 miR-663a 在胶质瘤细胞增殖、迁移、侵袭和癌症干细胞样 (CSC) 特性中的作用和潜在机制。采用定量逆转录 PCR (qRT-PCR) 检测胶质母细胞瘤组织和邻近正常组织中 miR-663a 的表达。此外，对 U-251 MG 细胞或人原发性胶质母细胞瘤癌细胞 (pGBMC1) 进行了 miR-633a 的功能增益和功能丧失测定。细胞增殖、迁移、侵袭、CSC 特性和干细胞标志物（包括 CD133、CD44）的谱分别通过 MTT 测定、Transwell 测定、肿瘤球实验和蛋白质印迹检查。进行双荧光素酶报告基因测定以证明 miR-663a 和赖氨酸去甲基化酶 2A (KDM2A) 之间的靶向关系。结果显示miR-663a在胶质母细胞瘤组织和细胞中下调。过表达 miR-663a 抑制了 U-251 MG 细胞和 pGBMC1 的增殖、迁移、侵袭和 CSC 特性，而 miR-663a 敲低具有相反的效果。这体内实验证实miR-663a抑制裸鼠U-251 MG细胞的生长。当与 THP1 细胞共培养时，U-251 MG 细胞获得了增强的增殖、迁移、侵袭和 CSC 特性。MiR-633a 过表达逆转了 THP1 介导的对 U-251 MG 细胞的影响，并降低了 THP1 细胞的“M2”极化。更重要的是，从机制上讲，KDM2A 被 miR-663a 靶向。KDM2A 敲低在体外抑制了 U-251 MG 细胞的进展和 CSC 特性，并抑制了 TGF-β。总体而言，这些数据表明，上调 miR-663a 通过抑制 KDM2A 介导的 TGF-β/Smad 通路来减少胶质瘤的进展。