Optometry Center, Shaanxi Eye Hospital, Xi'an People's Hospital (Xi'an Fourth Hospital), Affiliated Guangren Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi Province, China.
Department of Ophthalmology, The First People's Hospital of Chenzhou, Chenzhou, Hunan Province, China.
Bioengineered. 2022 Feb;13(2):3694-3706. doi: 10.1080/21655979.2021.2024977.
As a common intraocular malignancy in pediatrics, retinoblastoma (RB) has high prevalence worldwide. We conducted this study, aiming to explore the molecular mechanism of Krüppel-like transcription factor 16 (KLF16)/cellular retinoic acid-binding proteins-2 (CRABP2) in regulating the invasion and migration and apoptosis of RB cells via integrin-β1/focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) pathway. With the adoption of real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, the mRNA and protein expression of CRABP2 and KLF16 were measured. In addition, the proliferation, clone formation ability and migration were detected with methyl thiazolyl tetrazolium (MTT), clone formation and wound healing assays, respectively. Furthermore, the invasion and apoptosis of transfected WERI-RB1 cells were evaluated with transwell and Tunel assays. With the application of Western blot, the expressions of proliferation-, apoptosis- and pathway-related proteins were assayed. The combination of KLF16 and CRABP2 was confirmed by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). In this study, we found that CRABP2 gained a huge growth in RB cells and its silence promoted apoptosis but suppressed the proliferation, migration and invasiveness of WERI-RB1 cells. In addition, KLF16 could bind to CRABP2. It was also found that KLF16 overexpression reversed the effects of CRABP2 silence on the proliferation, migration and apoptosis of WERI-RB1 cells. What is more, CRABP2 silence blocked integrin-β1/FAK/ERK signaling pathway. In conclusion, KLF16 transcriptional up-regulation of CRABP2 promoted proliferation, invasion and migration but inhibited apoptosis of RB cells by activating integrin-β1/FAK/ERK pathway.
作为儿科常见的眼内恶性肿瘤，视网膜母细胞瘤（RB）在全球范围内发病率较高。本研究旨在通过整合素-β1/黏着斑激酶（FAK）/细胞外信号调节激酶（ERK）通路探讨 Krüppel 样转录因子 16（KLF16）/细胞视黄酸结合蛋白 2（CRABP2）在调节 RB 细胞侵袭、迁移和凋亡中的分子机制。采用实时定量聚合酶链反应（RT-qPCR）和 Western blot 检测 CRABP2 和 KLF16 的 mRNA 和蛋白表达。此外，采用甲基噻唑基四唑（MTT）、克隆形成和划痕愈合实验分别检测细胞增殖、克隆形成能力和迁移。进一步采用 Transwell 和 Tunel 实验检测转染 WERI-RB1 细胞的侵袭和凋亡。采用 Western blot 检测增殖、凋亡和通路相关蛋白的表达。通过双荧光素酶报告基因检测和染色质免疫沉淀（ChIP）实验证实 KLF16 与 CRABP2 的结合。本研究发现，CRABP2 在 RB 细胞中大量增殖，其沉默促进细胞凋亡，抑制 WERI-RB1 细胞的增殖、迁移和侵袭。此外，KLF16 可与 CRABP2 结合。还发现过表达 KLF16 可逆转 CRABP2 沉默对 WERI-RB1 细胞增殖、迁移和凋亡的影响。更重要的是，CRABP2 沉默阻断整合素-β1/FAK/ERK 信号通路。综上所述，KLF16 转录上调 CRABP2 通过激活整合素-β1/FAK/ERK 通路促进 RB 细胞增殖、侵袭和迁移，抑制细胞凋亡。
