circCSPP1调控miR-495-3p/XIAP轴对顺铂耐药骨肉瘤细胞增殖、迁移及侵袭的影响

Effect of circCSPP1 on the Proliferation, Migration and Invasion of Cisplatin-Resistant Osteosarcoma Cells by Regulating miR-495-3p/XIAP Axis

摘要

目的 探讨环状RNA中心体和纺锤体极相关蛋白1（circular RNA centrosome and spindle pole-associated protein 1，circCSPP1）调控miR-495-3p/X染色体连锁的凋亡抑制蛋白（X-linked inhibitor of apoptosis protein，XIAP）轴对顺铂耐药骨肉瘤细胞增殖、迁移及侵袭的影响。方法 采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测顺铂对骨肉瘤细胞（U2OS）、顺铂耐药骨肉瘤细胞（U2OS/DDP）的半数抑制浓度（half-maximal inhibitory concentration，IC₅₀）值。通过双荧光素酶报告实验、RNA免疫沉淀实验和RNA pull-down实验分析circCSPP1、miR-495-3p与XIAP的靶向关系。将U2OS/DDP细胞分为control组（未转染）、si-circCSPP1组（转染circCSPP1 siRNA）、si-NC组（转染siRNA阴性对照）、miR-NC组（转染模拟物阴性对照）、miR-495-3p mimic组（转染miR-495-3p模拟物）、si-circCSPP1 + miR-495-3p inhibitor组（转染circCSPP1 siRNA和miR-495-3p抑制物）、pcDNA-NC组（转染过表达阴性对照）、pcDNA-circCSPP1组（转染circCSPP1过表达载体质粒）。以实时荧光定量聚合酶链反应检测circCSPP1、miR-495-3p和XIAP mRNA表达；蛋白质免疫印迹检测XIAP蛋白表达；CCK-8法检测U2OS/DDP细胞存活率；集落形成实验检测U2OS/DDP细胞克隆能力；Transwell实验检测U2OS/DDP细胞迁移和侵袭情况。结果 第1、3、5代U2OS/DDP细胞的IC₅₀分别为23.44、20.38、17.38 μg∙mL^-1，显著高于U2OS细胞的3.17、3.06、2.79 μg∙mL^-1，前者分别是后者的7.39、6.66、6.23倍。circCSPP1与miR-495-3p存在直接相互作用，miR-495-3p与XIAP存在直接相互作用。相较于control组、si-NC组，si-circCSPP1组U2OS/DDP细胞高表达miR-495-3p（P < 0.05），XIAP mRNA和蛋白表达水平、细胞存活率、集落形成数、侵袭数、迁移数均降低（P < 0.05）。相较于control组、miR-NC组，miR-495-3p mimic组U2OS/DDP细胞XIAP mRNA和蛋白表达水平、细胞存活率、集落形成数、迁移数、侵袭数均降低（P < 0.05）。相较于si-circCSPP1组，si-circCSPP1 + miR-495-3p inhibitor组U2OS/DDP细胞XIAP mRNA和蛋白表达水平、细胞存活率、集落形成数、迁移数、侵袭数均升高（P < 0.05）。相较于control组、pcDNA-NC组，pcDNA-circCSPP1组U2OS/DDP细胞存活率、集落形成数、侵袭数、迁移数均升高（P < 0.05）。结论 干扰circCSPP1通过调控miR-495-3p/XIAP轴可抑制顺铂耐药骨肉瘤细胞增殖、迁移和侵袭。

Abstract

Objective To investigate the regulatory effect of circular RNA centrosome and spindle pole-associated protein 1 (circCSPP1) regulating miR-495-3p/X-linked inhibitor of apoptosis protein (XIAP) axis on the proliferation, migration and invasion of cisplatin-resistant osteosarcoma cells.Methods Cell counting kit-8 (CCK-8) method was used to detect the half-maximal inhibitory concentration (IC₅₀) value of cisplatin on osteosarcoma cells (U2OS) and cisplatin-resistant osteosarcoma cells (U2OS/DDP). Dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay were utilized to analyze the targeting relationship among circCSPP1, miR-495-3p, and XIAP . The U2OS/DDP cells were divided into control group (untransfected), si-circCSPP1 group (transfected with circCSPP1 siRNA), si-NC group (transfected with siRNA negative control), miR-NC group (transfected with mimic negative control), miR-495-3p mimic group (transfected with miR-495-3p mimic), si-circCSPP1 + miR-495-3p inhibitor group (transfected with circCSPP1 siRNA and miR-495-3p inhibitor), pcDNA-NC group (transfected with overexpression negative control), and pcDNA-circCSPP1 group (transfected with circCSPP1 overexpression plasmid). Subsequently, real-time quantitative polymerase chain reaction detected the expression of circCSPP1, miR-495-3p, and XIAP mRNA; while Western blot was used to detect XIAP protein expression; CCK-8 method was used to detect the survival rate of U2OS/DDP cells; colony formation assays was used to detect the cloning ability of U2OS/DDP cells. Transwell experiment was used to detect migration and invasion of U2OS/DDP cells.Results The IC₅₀ values for the 1st, 3rd and 5th generations of U2OS/DDP cells were 23.44, 20.38 and 17.38 μg∙mL^-1 respectively, which were significantly higher than those of U2OS cells (3.17, 3.06 and 2.79 μg∙mL^-1), and the former were 7.39, 6.66 and 6.23 times those of the latter. Mechanistically, circCSPP1 directly interacted with miR-495-3p, and miR-495-3p directly interacted with XIAP. Compared with the control group and the si-NC group, miR-495-3p expression of U2OS/DDP cells in the si-circCSPP1 group was significantly increased (P < 0.05), and XIAP mRNA and protein expression, cell survival rate, colony formation number, migration number and invasion number were significantly reduced (P < 0.05). Compared with the control group and the miR-NC group, the XIAP mRNA and protein expression, cell survival rate, colony formation number, invasion number and migration number of U2OS/DDP cells in the miR-495-3p mimic group were significantly reduced (P < 0.05). compared with the si-circCSPP1 group, the XIAP mRNA and protein expression, cell survival rate, colony formation number, invasion number and migration number of U2OS/DDP cells in the si-circCSPP1 + miR-495-3p inhibitor group were significantly increased (P < 0.05). Furthermore, compared with the control group and the pcDNA-NC group, the survival rate, colony formation number, invasion number and migration number of U2OS/DDP cells in the pcDNA-circCSPP1 group were all increased (P < 0.05). Conclusion Interfering with circCSPP1 could inhibit the proliferation, migration and invasion of cisplatin-resistant osteosarcoma cells by regulating the miR-495-3p/XIAP axis.

关键词

顺铂 / 骨肉瘤 / 环状RNA中心体和纺锤体极相关蛋白1 / 微小RNA-495-3p / X染色体连锁的凋亡抑制蛋白 / 耐药

Key words

cisplatin / osteosarcoma / circular RNA centrosome and spindle pole-associated protein 1 / miR-495-3p / X-linked inhibitor of apoptosis protein / drug resistance

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