circFAT1靶向miR-145调控骨肉瘤细胞增殖和凋亡的作用及机制  

Effect and mechanism of circFAT1 targeting miR-145 in regulating proliferation and apoptosis of osteosarcoma cells

作　　者：刘艳武 王玲娟 赵轶男 霍军丽 LIU Yanwu;WANG Lingjuan;ZHAO Yinan;HUO Junli(First Affiliated Hospital of PLA Air Force Military Medical University,Xi'an,Shaanxi 710032,China)

机构地区：[1]中国人民解放军空军军医大学第一附属医院骨科,陕西西安710032 [2]中国人民解放军空军军医大学第一附属医院神经外科,陕西西安710032

出　　处：《中华肿瘤防治杂志》2024年第23期1429-1436,共8页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的探讨circFAT1调控miR-145对骨肉瘤细胞增殖和凋亡的作用及其潜在机制,为骨肉瘤的分子靶向治疗提供新的视角和理论依据。方法收集2015-07-10-2017-02-10在空军军医大学第一附属医院骨科住院治疗的30例骨肉瘤患者的骨肉瘤组织和癌旁正常组织,采用实时荧光定量聚合酶链式反应(qRT-PCR)法检测circFAT1和miR-145在骨肉瘤组织和细胞中的表达水平。在HOS和U2OS细胞中分别转染sh-NC和sh-circFAT1后,采用细胞计数试剂盒8(CCK8)法检测细胞增殖情况,qRT-PCR法检测增殖相关分子Ki-67表达水平,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测凋亡相关分子cleaved-Caspase-3的表达水平。采用双荧光素酶报告基因和RNA结合蛋白免疫沉淀(RIP)法检测circFAT1和miR-145结合情况。采用Pearson相关分析探讨circFAT1与miR-145在骨肉瘤组织中表达的相关关系。运用Kaplan-Meier法绘制生存曲线,并通过log-rank检验评估生存率的差异。结果circFAT1在骨肉瘤组织(2.00±0.56 vs 1.21±0.40,t=6.094,P<0.001)和细胞(HOS:1.42±0.11,U2OS:2.36±0.20;vs Host:1.00±0.09,t值分别为5.174和11.030,均P<0.001)中高表达并与患者预后不良(χ^(2)=4.264,P=0.039)有关。沉默circFAT1能够抑制HOS(F=11.423,P=0.003)和U2OS(F=8.004,P=0.009)细胞增殖和下调增殖相关分子Ki-67(HOS:1.00±0.05 vs 0.65±0.05,t=8.156,P=0.001;U2OS:1.00±0.03 vs 0.55±0.06,t=11.186,P<0.001)的表达水平;过表达circFAT1促进HOS(5.50±0.56 vs 10.23±1.03,t=7.006,P=0.002)和U2OS(6.25±0.64 vs 12.02±1.10,t=7.855,P=0.001)细胞凋亡和上调凋亡相关分子cleaved-Caspase-3(HOS:1.00±0.04 vs 1.36±0.12,t=4.789,P=0.009;U2OS:1.00±0.04 vs 1.45±0.13,t=5.721,P=0.005)的表达水平。荧光素酶报告基因和RIP检测结果显示,circFAT1能够通过ceRNA机制靶向结合miR-145。沉默circFAT1上调HOS(1.00±0.04 vs 1.52±0.11,t=7.850,P=0.001)和U2OS(1.00±0.05 vs 1.64±0.12,t=8.648,P=0.001)细胞中miR-145表达水平。miR-145在骨肉瘤组织Objective To investigate the effect and mechanism of circFAT1 regulating miR-145 on proliferation and apoptosis of osteosarcoma cells,providing a new perspective and theoretical basis for molecular targeted therapy of osteosarcoma.Methods Osteosarcoma tissues and adjacent normal tissues were collected from 30 patients who were treated in the Department of Orthopedics at Xijing Hospital,Air Force Medical University,between July 10,2015,and February 10,2017.The expression levels of circFAT1 and miR-145 in osteosarcoma tissues and cells were detected by quantitative real-time polymerase chain reaction(qRT-PCR).After transfecting HOS and U2OS cells with sh-NC and sh-circFAT1,respectively,cell proliferation was detected by using the cell counting kit-8(CCK8)assay,and the expression level of the proliferation-related molecule Ki-67 was measured by qRT-PCR.Cell apoptosis was assessed by flow cytometry,and the expression level of the apoptosis-related molecule cleaved-Caspase-3 was determined by Western blot analysis.The binding of circFATl and miR-145 was detected using dual-luciferase reporter and RNA immunoprecipitation(RIP)assays.Pearson correlation analysis was used to investigate the correlation between circFATl and miR-145 expression in osteosarcoma tissues.Survival curves were plotted using the Kaplan-Meier method,and the differences in survival rates were evaluated by using the log-rank test.Results CircFAT1 was highly expressed in osteosarcoma tissues(2.00±0.56 vs1.21±0.40,t=6.094,P<0.001)and cells(HOS:1.42±0.11,U2OS:2.36±0.20;vs Host:1.00±0.09,t values were 5.174 and 11.030,both P<0.001)and was associated with poor prognosis(χ^(2)=4.264,P=0.039),Silencing circFAT1 inhibited the proliferation of HOS(F=11.423,P=0.003)and U2OS(F=8.004,P=0.009)cells and downregulated the expression of the proliferation-related molecule Ki-67(HOS:1.00±0.05 vs 0.65±0.05,t=8.156,P=0.001;U20S:1.00±0.03 vs 0.55±0.06,t=11.186,P<0.001).Overexpression of circFAT1 promoted apoptosis in HOS(5.50±0.56 vs 10.23±1.03,t=7.006,P=0.00

关 键 词：骨肉瘤 circFAT1 MIR-145 增殖 凋亡 

分 类 号：R738.1[医药卫生—肿瘤]