Department of Orthopedics, Shaoxing Shangyu People's Hospital, No. 517 Civic Avenue, Baiguan Street, Shangyu District, Shaoxing, Zhejiang, 312300, China.
J Orthop Surg Res. 2021 Jan 13;16(1):50. doi: 10.1186/s13018-020-02169-5.
Circular RNAs (circRNAs) have been shown to participate in the chemoresistance and tumorigenesis of multiple cancers. The purpose of this research was to investigate the function of circ_0081001 in methotrexate (MTX) resistance of osteosarcoma (OS) and its potential molecular mechanism.
The expression of circ_0081001, cytochrome P450 family 51 subfamily A member 1 (CYP51A1), and miR-494-3p was detected by qRT-PCR. Cell viability, apoptosis, migration, and invasion were evaluated by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and transwell assay, respectively. Western blot (WB) assay was used to measure the protein levels of cleaved-caspase3 (cleaved-casp3), E-cadherin, N-cadherin, and transglutaminase-2 (TGM2). The interaction between miR-494-3p and circ_0081001 or TGM2 was predicted by bioinformatics analysis and verified using the dual-luciferase reporter assay. The mice xenograft model was established to investigate the roles of circ_0081001 in MTX resistance of OS in vivo.
Circ_0081001 and TGM2 were upregulated, and miR-494-3p was downregulated in MTX-resistant OS tissues and cells. Moreover, circ_0081001 interference enhanced cell sensitivity to MTX through promoting apoptosis and inhibiting cell viability and metastasis in vitro. Furthermore, circ_0081001 was identified as a molecular sponge of miR-494-3p to upregulate TGM2 level. In addition, circ_0081001 knockdown inhibited MTX resistance via upregulating miR-494-3p and downregulating TGM2. Besides, circ_0081001 downregulation improved MTX sensitivity of OS in vivo.
Knockdown of circ_0081001 enhanced MTX sensitivity of OS cells through downregulating TGM2 by sponging miR-494-3p, elucidating a novel regulatory mechanism for chemoresistance of OS and providing a potential circRNA-targeted therapy for OS.
环状 RNA（circRNA）已被证明参与多种癌症的化疗耐药和肿瘤发生。本研究旨在探讨 circ_0081001 在骨肉瘤（OS）中甲氨蝶呤（MTX）耐药中的作用及其潜在的分子机制。
通过 qRT-PCR 检测 circ_0081001、细胞色素 P450 家族 51 亚家族 A 成员 1（CYP51A1）和 miR-494-3p 的表达。通过细胞计数试剂盒-8（CCK-8）测定、流式细胞术和 Transwell 测定分别评估细胞活力、凋亡、迁移和侵袭。Western blot（WB）测定用于测量裂解型胱天蛋白酶 3（cleaved-caspase3）、E-钙黏蛋白、N-钙黏蛋白和转谷氨酰胺酶 2（TGM2）的蛋白水平。通过生物信息学分析预测 miR-494-3p 与 circ_0081001 或 TGM2 的相互作用，并通过双荧光素酶报告基因测定进行验证。建立小鼠异种移植模型以研究 circ_0081001 在体内对 OS 的 MTX 耐药作用。
MTX 耐药的 OS 组织和细胞中 circ_0081001 和 TGM2 上调，miR-494-3p 下调。此外，circ_0081001 干扰通过促进细胞凋亡和抑制体外细胞活力和转移来增强细胞对 MTX 的敏感性。此外，circ_0081001 被鉴定为 miR-494-3p 的分子海绵，以上调 TGM2 水平。此外，circ_0081001 下调通过上调 miR-494-3p 和下调 TGM2 抑制 OS 的 MTX 耐药性。此外，circ_0081001 下调在体内改善了 OS 的 MTX 敏感性。
通过海绵吸附 miR-494-3p 下调 TGM2，circ_0081001 敲低增强了 OS 细胞对 MTX 的敏感性，揭示了 OS 化疗耐药的新调控机制，并为 OS 提供了一种潜在的环状 RNA 靶向治疗方法。
