Department of Ophthalmology, The First Affiliated Hospital of Chengdu Medical College, Chengdu, China.
Department of Ophthalmology, Nuclear Industry 416th Hospital, Chengdu, China.
Bioengineered. 2022 Mar;13(3):5638-5652. doi: 10.1080/21655979.2022.2033464.
The oncogenic role of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) has been reported in retinoblastoma (RB). However, the underlying regulatory mechanisms remain poorly understood. In this study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were performed to analyze the expression of AFAP1-AS1, microRNA miR-545-3p, or G protein subunit beta 1 (GNB1). Cell Counting Kit-8 (CCK-8) and Transwell migration assays were used to detect cell proliferation and migration. In addition, caspase-3 activity was monitored by caspase-3 activity assay. Luciferase reporter assays combined with RNA immunoprecipitation (RIP) and pull-down assays were performed to elucidate the target relationship between miR-545-3p and AFAP1-AS1 or GNB1. Xenograft tumor experiments were performed to evaluate RB cell growth . Increased AFAP1-AS1 and GNB1 expression in RB tissues and cells was confirmed by RT-qPCR; conversely, miR-545-3p was found to be downregulated in RB tissues and cells. AFAP1-AS1 overexpression resulted in increased proliferation and migration of RB cells, whereas AFAP1-AS1 silencing resulted in decreased proliferation and migration of RB cells. Moreover, AFAP1-AS1 was found to target miR-545-3p. The anti-miR-545-3p treatment phenocopied the effect of AFAP1-AS1 overexpression and promoted RB cell growth . miR-545-3p was found to directly target GNB1. GNB1 silencing resulted in reduced proliferation and migration of RB cells and attenuated the oncogenic effect of the miR-545-3p inhibitor. Thus, in this study, a novel ceRNA regulation network of AFAP1-AS1 in RB was identified, where AFAP1-AS1 regulated GNB1 expression by targeting miR-545-3p, ultimately driving RB progression.
AFAP1-AS1 在视网膜母细胞瘤（RB）中具有致癌作用。然而，其潜在的调控机制仍知之甚少。本研究采用逆转录定量聚合酶链反应（RT-qPCR）和 Western blot 分析 AFAP1-AS1、微小 RNA miR-545-3p 或 G 蛋白亚基β1（GNB1）的表达。细胞计数试剂盒-8（CCK-8）和 Transwell 迁移实验检测细胞增殖和迁移。此外，通过 caspase-3 活性测定检测 caspase-3 活性。通过荧光素酶报告基因实验结合 RNA 免疫沉淀（RIP）和下拉实验阐明 miR-545-3p 与 AFAP1-AS1 或 GNB1 的靶标关系。进行异种移植肿瘤实验以评估 RB 细胞生长。通过 RT-qPCR 证实 RB 组织和细胞中 AFAP1-AS1 和 GNB1 表达增加，而 miR-545-3p 在 RB 组织和细胞中表达下调。AFAP1-AS1 过表达导致 RB 细胞增殖和迁移增加，而 AFAP1-AS1 沉默导致 RB 细胞增殖和迁移减少。此外，发现 AFAP1-AS1 靶向 miR-545-3p。抗 miR-545-3p 处理模拟了 AFAP1-AS1 过表达的作用，并促进了 RB 细胞生长。发现 miR-545-3p 直接靶向 GNB1。GNB1 沉默导致 RB 细胞增殖和迁移减少，并减弱 miR-545-3p 抑制剂的致癌作用。因此，在本研究中，鉴定了 RB 中 AFAP1-AS1 的新型 ceRNA 调控网络，其中 AFAP1-AS1 通过靶向 miR-545-3p 调节 GNB1 表达，最终驱动 RB 进展。
