Department of Strabismus and Pediatric Ophthalmology, Shanxi Provincial Eye Hospital, Taiyuan, Shanxi, China (mainland).
Department of Orbit and Ocular Tumor, Shanxi Provincial Eye Hospital, Taiyuan, Shanxi, China (mainland).
Med Sci Monit. 2020 Jul 9;26:e923817. doi: 10.12659/MSM.923817.
BACKGROUND Retinoblastoma (RB) seriously endangers the vision and even the life of patients. This study aimed to explore the endoplasmic reticulum stress (ERS) and drug resistance of RB and verify the effect of miR-512-3p as a regulator of XBP-1 shearing mechanism on ERS, proliferation, apoptosis, and autophagy levels of RB cells. MATERIAL AND METHODS Y79, weri-RB1, and HXO-RB44 cells were treated with cisplatin (DDP) gradient concentration to construct DDP-resistant cells. The drug inhibition rate and cell proliferation were assessed by CCK-8 assay. The cell transfection and cell apoptosis were detected by RT-qPCR analysis and TUNEL assay, respectively. The protein expression was detected by Western blot analysis. Dual-luciferase reporter assay confirmed the combination of miR-512-30p and XBP-1 transcript 1/2. RESULTS DDP inhibition rates for DDP-resistant RB cells were lower than that for RB cells. The XBP-1 expression was increased in DDP-resistant RB cells, and Y79 cells were chosen for the subsequent experiments. After transfection, miR-512-3p overexpression obviously inhibited the proliferation of DDP-resistant Y79 cells (Y79/DDP cells). miR-512-3p overexpression increased the DDP inhibition rate for Y79/DDP cells and apoptosis of Y79/DDP cells. miR-512-3p overexpression downregulated the expression of LC3 II/I in Y79/DDP cells. The effect of miR-512-3p inhibition on Y79/DDP cells was not as obvious as the effect of miR-512-3p overexpression on Y79/DDP cells. Furthermore, miR-512-3p was confirmed to be combined with XBP-1 transcript variant 1. CONCLUSIONS miR-512-3p improved the DDP resistance of RB cells by promoting ERS-induced apoptosis and inhibiting the proliferation and autophagy of RB cells.
视网膜母细胞瘤(RB)严重威胁患者的视力甚至生命。本研究旨在探讨内质网应激(ERS)与 RB 耐药性的关系,并验证 miR-512-3p 作为 XBP-1 剪接机制调节剂对 RB 细胞 ERS、增殖、凋亡和自噬水平的影响。
用顺铂(DDP)梯度浓度处理 Y79、weri-RB1 和 HXO-RB44 细胞,构建 DDP 耐药细胞。通过 CCK-8 检测药物抑制率和细胞增殖。分别通过 RT-qPCR 分析和 TUNEL 检测细胞转染和细胞凋亡。通过 Western blot 分析检测蛋白表达。双荧光素酶报告实验证实了 miR-512-3p 与 XBP-1 转录本 1/2 的结合。
DDP 耐药 RB 细胞的 DDP 抑制率低于 RB 细胞。DDP 耐药 RB 细胞中 XBP-1 表达增加,选择 Y79 细胞进行后续实验。转染后,miR-512-3p 过表达明显抑制 DDP 耐药 Y79 细胞(Y79/DDP 细胞)的增殖。miR-512-3p 过表达增加了 Y79/DDP 细胞的 DDP 抑制率和细胞凋亡。miR-512-3p 过表达下调了 Y79/DDP 细胞中 LC3 II/I 的表达。miR-512-3p 抑制对 Y79/DDP 细胞的作用不如 miR-512-3p 过表达对 Y79/DDP 细胞的作用明显。此外,证实 miR-512-3p 与 XBP-1 转录本变体 1 结合。
miR-512-3p 通过促进 ERS 诱导的凋亡和抑制 RB 细胞的增殖和自噬来提高 RB 细胞对 DDP 的耐药性。