Department of Medical Administration, Shanghai Pudong Hospital, Shanghai, China.
Department of Ophthalmology, Shanghai Pudong Hospital, Shanghai, China.
Neurochem Res. 2020 Nov;45(11):2691-2702. doi: 10.1007/s11064-020-03112-0. Epub 2020 Aug 31.
Retinoblastoma (RB) is a common intraocular malignant tumor. The growing evidence has reported that circular RNAs (circRNAs) play critical roles in RB development. Therefore, the purpose of the study is to investigate the regulatory mechanism of circDHDDS in RB. The real-time quantitative polymerase chain reaction (RT-qPCR) assay was used to quantify the expression levels of circDHDDS, miR-361-3p, and WNT3A in RB tissues and cells (RPCs, Y-79, and WERI-Rb-1). The proliferation and cell cycle of RB cells were assessed by colony formation assay and flow cytometry assays, respectively. The migration and invasion of RB cells were measured by transwell assay. The protein expression levels of Nectin-3 (CD113), SOX2, Nanog, and WNT3A were measured by Western blot assay. The functional targets of circDHDDS and miR-361-3p were predicted by bioinformatics databases, and the dual-luciferase reporter assay was used to confirm the interaction relationship between miR-361-3p and circDHDDS or WNT3A. The functional role of circDHDDS silencing in vivo was evaluated by xenograft experiment. We found that circDHDDS was overexpressed in RB tissues and cells compared with normal retinas tissues and retinal pigment epithelial cells, correspondingly. Furthermore, silencing of circDHDDS impeded proliferation, migration, invasion, and induced cell cycle arrest in vitro, which were abolished by knockdown of miR-361-3p. The in vivo experiments also suggested that tumor growth was inhibited by knockdown of circDHDDS. Moreover, we also found that miR-361-3p specifically bound to WNT3A, and overexpression of miR-361-3p suppressed RB development by decreasing WNT3A expression. Summarily, circDHDDS, a molecule sponge of miR-361-3p, regulated the expression of WNT3A. Therefore, circDHDDS/miR-361-3p/WNT3A axis stimulated the development of RB by regulation of proliferation, cell cycle program, migration, and invasion of RB cells.
视网膜母细胞瘤（RB）是一种常见的眼内恶性肿瘤。越来越多的证据表明，环状 RNA（circRNA）在 RB 发展中发挥着关键作用。因此，本研究旨在探讨 circDHDDS 在 RB 中的调控机制。实时定量聚合酶链反应（RT-qPCR）检测 RB 组织和细胞（RPCs、Y-79 和 WERI-Rb-1）中 circDHDDS、miR-361-3p 和 WNT3A 的表达水平。通过集落形成实验和流式细胞术分别评估 RB 细胞的增殖和细胞周期。通过 Transwell 实验测量 RB 细胞的迁移和侵袭。通过 Western blot 实验测量 Nectin-3（CD113）、SOX2、Nanog 和 WNT3A 的蛋白表达水平。通过生物信息学数据库预测 circDHDDS 和 miR-361-3p 的功能靶标，并通过双荧光素酶报告基因实验证实 miR-361-3p 与 circDHDDS 或 WNT3A 的相互作用关系。通过异种移植实验评估 circDHDDS 沉默在体内的功能作用。我们发现与正常视网膜组织和视网膜色素上皮细胞相比，circDHDDS 在 RB 组织和细胞中过度表达。此外，体外沉默 circDHDDS 可抑制增殖、迁移和侵袭，并诱导细胞周期停滞，而 miR-361-3p 的敲低可消除这些作用。体内实验也表明，circDHDDS 的敲低抑制了肿瘤的生长。此外，我们还发现 miR-361-3p 特异性结合 WNT3A，而过表达 miR-361-3p 通过降低 WNT3A 的表达抑制 RB 的发展。总之，circDHDDS 作为 miR-361-3p 的分子海绵，通过调节 WNT3A 的表达来调节 RB 的发展。因此，circDHDDS/miR-361-3p/WNT3A 轴通过调节 RB 细胞的增殖、细胞周期进程、迁移和侵袭来刺激 RB 的发展。
