lncRNA FGD5-AS1调控miR-761影响骨肉瘤细胞增殖、迁移和凋亡的机制研究

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摘要目的探讨长链非编码RNA(long non-coding RNA，lncRNA)中含有5个PH结构域的反义RNA1(PH domain containing 5 antisense RNA 1，FGD5-AS1)和可能机制对骨肉瘤细胞增殖、迁移和凋亡的影响。方法收集2016—2018年在本院接受手术的30例骨肉瘤患者的骨肉瘤组织和瘤旁组织标本，男18例，女12例；年龄7~69岁，平均年龄(20.2±1.29)岁。采用逆转录定量聚合酶链式反应(reverse transcription-quantitative polymerase chain reaction，RT-qPCR)检测FGD5-AS1的表达水平。将骨肉瘤细胞U2OS分为转染小干扰RNA阴性对照(si-NC)组、转染si-FGD5-AS1(si-FGD5-AS1)组、转染si-FGD5-AS1和miRNA模拟物阴性对照(si-FGD5-AS1+miR-NC)组、转染si-FGD5-AS1和miR-761 mimics(si-FGD5-AS1+miR-761)组、转染si-FGD5-AS1和miRNA抑制物阴性对照(si-FGD5-AS1和anti-miR-NC)组和转染si-FGD5-AS1和miR-761抑制物(si-FGD5-AS1和anti-miR-761)组。噻唑蓝(methyl thiazolyltetrazolium，MTT)法、集落形成实验、流式细胞术分别检测细胞活力、克隆形成能力和细胞凋亡。双荧光素酶报告实验和RT-qPCR确定FGD5-AS1对miR-761的靶向调控作用。结果与瘤旁组织比较，骨肉瘤组织中FGD5-AS1的表达水平显著升高(P＜0.05)。与si-NC组比较，si-FGD5-AS1组U2OS细胞存活率、克隆形成数、迁移细胞数显著降低，细胞凋亡率显著升高(P＜0.05)。与si-FGD5-AS1+miR-NC组比较，si-FGD5-AS1+miR-761组U2OS细胞存活率、克隆形成数、迁移细胞数显著降低，细胞凋亡率显著升高(P＜0.05)。与si-FGD5-AS1和anti-miR-NC组比较，si-FGD5-AS1和anti-miR-761组U2OS细胞存活率、克隆形成数、迁移细胞数显著升高，而细胞凋亡率显著降低(P＜0.05)。FGD5-AS1对miR-761具有靶向负性调控作用。结论骨肉瘤中FGD5-AS1呈高表达。抑制FGD5-AS1通过上调miR-761可抑制骨肉瘤细胞增殖和迁移，诱导细胞凋亡。

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	李锋
	龚继承
	杜经柱
	陈奇

关键词 ： FGD5-AS1, miR-761, 骨肉瘤, 增殖, 迁移, 细胞凋亡

Abstract：Objective To investigate the effect of long-chain non-coding RNA(lncRNA) PH domain containing 5 antisense RNA 1(FGD5-AS1) on the proliferation，migration and apoptosis of osteosarcoma cells by targeting miR-761.Methods Osteosarcoma tissue and paratumoral tissue samples were collected from 30 patients with osteosarcoma，who underwent surgery in our hospital from 2016 to 2018.There were male 18 and female 12，age 7~69 years old，mean age (20.2±1.29) years old.The expression level of FGD5-AS1 was detected by real-time fluorescent quantitative PCR (RT-qPCR).Osteosarcoma cells U2OS were divided into transfection small interfering RNA negative control (si-NC) group，transfection of si-FGD5-AS1 (si-FGD5-AS1) group，transfection of si-FGD5-AS1 and miRNA mimics negative control (si-FGD5-AS1+miR-NC) group，transfection of si-FGD5-AS1 plus miR-761 mimics (si-FGD5-AS1+miR-761) group，transfection of si-FGD5-AS1 plusmiRNA inhibitor negative control (si-FGD5-AS1+anti-miR-NC) group and transfection of si-FGD5-AS1 plus miR-761 inhibitors (si-FGD5-AS1+anti-miR-761) group.The methyl thiazolyl tetrazolium (MTT) method，colony formation experiment，and flow cytometry were used to detect cell viability，colony forming ability，and apoptosis，respectively.Double luciferase reporting experiments and RT-qPCR confirmed the targeted regulation of FGD5-AS1 on miR-761.Results Compared with adjacent tumor tissues，the expression level of FGD5-AS1 in osteosarcoma tissues was significantly increased (P＜0.05).Compared with the si-NC group，the survival rate，clone formation numbers，migrating cell numbers of U2OS cells in the si-FGD5-AS1 group was significantly reduced，while cell apoptosis rate was significantly increased (P＜0.05).Compared with the si-FGD5-AS1+miR-NC group，the survival rate，clone formation numbers，migrating cell numbers of U2OS cells in the si-FGD5-AS1+miR-761 group was significantly reduced，while cell apoptosis rate was significantly increased (P＜0.05).Compared with the si-FGD5-AS1+anti-miR-NC group，the survival rate，clone formation numbers，migrating cell numbers of U2OS cells in the si-FGD5-AS1+anti-miR-761 group was significantly increased，while cell apoptosis rate was significantly reduced (P＜0.05).FGD5-AS1 has a targeted negative regulatory effect on miR-761.Conclusion FGD5-AS1 is highly expressed in osteosarcoma.Inhibiting FGD5-AS1 inhibits the proliferation and migration，and induce apoptosis of osteosarcoma cells by up-regulating miR-761.

Key words： FGD5-AS1 miR-761 osteosarcoma proliferation migration apoptosis

作者简介: 李锋(1984－ )，男，主治医师，中国人民解放军联勤保障部队第九二八医院骨科，570206。

引用本文:

李锋，龚继承，杜经柱，陈奇. lncRNA FGD5-AS1调控miR-761影响骨肉瘤细胞增殖、迁移和凋亡的机制研究[J]. 实用骨科杂志, 2021, 27(3): 228-234.
Li Feng，Gong Jicheng，Du Jingzhu，et al. FGD5-AS1 Regulating miR-761 Affecting the Proliferation，Migration and Apoptosis of Osteosarcoma Cells. sygkzz, 2021, 27(3): 228-234.

链接本文:

http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2021/V27/I3/228

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