沉默环状 RNA hsa_circABCC1 通过海绵吸附 miR-591 下调 HDAC4 抑制骨肉瘤进展。

Department of Orthopedics, Second Hospital of Lanzhou University, Lanzhou, China.

Department of Oncology, Second Hospital of Lanzhou University, Lanzhou, China.

Environ Toxicol. 2023 Jul;38(7):1565-1576. doi: 10.1002/tox.23786. Epub 2023 Apr 13.

BACKGROUND

Circular RNA (circRNA) has been shown to play an important regulatory role in the development of various cancers, including osteosarcoma (OS). However, the role of circRNA ABCC1 (circABCC1) in OS was still poorly understood. The aim of our study was to investigate the role of circABCC1 in OS progression and its potential molecular mechanisms.

METHODS

The expression of circABCC1, microRNA-591 (miR-591) and histone deacetylase 4 (HDAC4) in OS tissues or cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) analyses. In vitro experiments, the viability, proliferation, apoptosis, migration, invasion and autophagy of U2OS and HOS cells were assessed in vitro using cell counting kit-8 (CCK-8) assay, 5-ethynyl-29-deoxyuridine (EdU) assay, flow cytometry (FCM) assay, transwell migration and invasion assays (transwell) and WB assay, respectively. Interactions between circABCC1 and miR-591, miR-591 and HDAC4 were confirmed using a dual luciferase reporter gene assay system. The oncogenic role of circABCC1 in OS in vivo was examined by establishing a tumor xenograft model.

RESULTS

CircABCC1 was significantly elevated in OS tissues (about 3.1-folds) and cells (U2OS (about 2.1-folds) and HOS (about 2.8-folds)) compared with the control (p < .05). Silencing of circABCC1 significantly reduced the viability and proliferation, promoted apoptosis, impaired migration and invasion, and increased autophagy of U2OS and HOS cells (p < .05). In addition, miR-591 was confirmed to be a target of circABCC1, exerting an opposite effect to circABCC1 (p < .05). MiR-591 attenuation in U2OS and HOS cells was able to reply to the inhibition of cell proliferation, migration and invasion as well as promotion of cell apoptosis and autophagy mediated by silencing circABCC1 (p < .05). HDAC4 was verified to be the target gene of miR-591 in U2OS and HOS cells and was regulated by the circABCC1/miR-591 axis (p < .05), and restoration of HDAC4 levels in U2OS and HOS cells was able to restore the altered cellular function caused by silencing circABCC1 (p < .05). In addition, knockdown of circABCC1 attenuated tumor growth in vivo (p < .05).

CONCLUSION

Silencing of circABCC1 inhibits osteosarcoma progression by attenuating HDAC4 expression through sponging miR-591.

背景

环状 RNA（circRNA）已被证明在各种癌症的发展中发挥重要的调控作用，包括骨肉瘤（OS）。然而，circABCC1 在 OS 中的作用仍知之甚少。本研究旨在探讨 circABCC1 在 OS 进展中的作用及其潜在的分子机制。

方法

采用实时定量聚合酶链反应（qRT-PCR）和 Western blot（WB）分析检测 OS 组织或细胞中 circABCC1、微小 RNA-591（miR-591）和组蛋白去乙酰化酶 4（HDAC4）的表达。在体外实验中，通过细胞计数试剂盒-8（CCK-8）测定、5-乙炔基-2'-脱氧尿苷（EdU）测定、流式细胞术（FCM）测定、Transwell 迁移和侵袭测定（Transwell）和 WB 测定，分别评估 U2OS 和 HOS 细胞的活力、增殖、凋亡、迁移、侵袭和自噬。采用双荧光素酶报告基因检测系统证实 circABCC1 与 miR-591、miR-591 与 HDAC4 之间的相互作用。通过建立肿瘤异种移植模型，检测 circABCC1 在体内对 OS 的致癌作用。

结果

与对照组相比，OS 组织（约 3.1 倍）和细胞（U2OS（约 2.1 倍）和 HOS（约 2.8 倍）中 circABCC1 明显升高（p < 0.05）。沉默 circABCC1 可显著降低 U2OS 和 HOS 细胞的活力和增殖，促进细胞凋亡，损害细胞迁移和侵袭，增加细胞自噬（p < 0.05）。此外，证实 miR-591 是 circABCC1 的靶基因，发挥与 circABCC1 相反的作用（p < 0.05）。在 U2OS 和 HOS 细胞中，miR-591 的衰减能够回复由沉默 circABCC1 介导的细胞增殖、迁移和侵袭抑制以及细胞凋亡和自噬促进（p < 0.05）。在 U2OS 和 HOS 细胞中，HDAC4 被验证为 miR-591 的靶基因，并受 circABCC1/miR-591 轴的调控（p < 0.05），在 U2OS 和 HOS 细胞中恢复 HDAC4 水平能够恢复沉默 circABCC1 引起的细胞功能改变（p < 0.05）。此外，沉默 circABCC1 可减弱体内肿瘤生长（p < 0.05）。