lncRNA PVT1和微小RNA-106a-5p对骨肉瘤MG-63细胞的影响及机制研究  

Investigation of the impacts of lncRNA PVT1 and microRNA-106a-5p in osteosarcoma MG-63 cells and its mechanisms

作　　者：魏建仝 张晓云 邹永刚 王勇平[3] WEI Jian-tong;ZHANG Xiao-yun;ZOU Yong-gang;WANG Yong-ping(Department of Orthopedics,Zhangye People's Hospital Affiliated to Hexi University,Zhangye 734000,China;Department of Pathology,Zhangye People's Hospital Affiliated to Hexi University,Zhangye 734000,China;Department of Orthopedics,The First Hospital of Lanzhou University,Lanzhou 730000,China)

机构地区：[1]河西学院附属张掖人民医院骨科,甘肃张掖734000 [2]河西学院附属张掖人民医院病理科,甘肃张掖734000 [3]兰州大学第一医院骨科,甘肃兰州730000

出　　处：《哈尔滨医科大学学报》2024年第3期227-231,共5页Journal of Harbin Medical University

基　　金：甘肃省自然科学基金资助项目(22JR5RG570);河西学院校长基金青年项目(QN2022006);兰州市人才创新创业项目(2021-RC-114);兰州大学第一医院科研计划项目(LDYYYN2021-12)。

摘　　要：目的探讨长链非编码RNA(long non-coding RNA,lncRNA)PVT1和miR-106a-5p对骨肉瘤细胞恶性行为的影响及分子机制。方法将骨肉瘤MG-63细胞分为NC组、PVT1-siRNA组、miRNA抑制剂组以及共转染组。以CCK-8实验检测细胞增殖能力,以流式细胞仪检测细胞凋亡情况,以DCFH-DA法检测细胞活性氧(reactive oxygen species,ROS)水平,以实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测基因表达,以蛋白印迹法检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4),铁蛋白(ferritin heavy chain,FTH)、血红素氧合酶1(heme oxygenase-1,HO1)和MDM4(murine double minute 4,MDM4)的表达水平。结果CCK-8实验结果显示敲低lncRNA PVT1后MG-63细胞12 h、24 h、48 h、72 h增殖能力降低(P<0.05),流式细胞仪检测结果显示敲低lncRNA PVT1后MG-63细胞凋亡水平以及ROS水平显著升高(P<0.05),lncRNA PVT1敲低联合miR-106a-5p抑制剂相对于lncRNA PVT1敲低组结果逆转(P<0.05)。RT-qPCR实验结果显示敲低lncRNA PVT1后miR-106a-5p基因表达水平升高并抑制MDM4基因表达(P<0.05),Western blot结果显示敲低lncRNA PVT1后抑制MDM4、HO1、GPX4和FTH蛋白水平(P<0.05)。结论在人骨肉瘤MG-63中,抑制lncRNA PVT1表达可促进miR-106a-5p表达,促进铁死亡和凋亡并抑制增殖。Objective To investigate the effects of lncRNA PVT1 and miR-106a-5p on the malignant behaviors of osteosarcoma cells and its molecular mechanisms.Methods Osteosarcoma MG-63 cells were divided into NC group,PVT1-siRNA group,miRNA inhibitor group,and co-transfection group.Cell proliferation ability was detected by CCK-8 assay,cell apoptosis was detected by flow cytometry,cellular reactive oxygen species(ROS)levels were measured by DCFH-DA method,gene expression was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),and the expression levels of glutathione peroxidase 4(GPX4),ferritin heavy chain(FTH),heme oxygenase-1(HO1),and murine double minute 4(MDM4)were detected by Western blot.Results The results of CCK-8 assay showed that the proliferative capacity of MG-63 cells was reduced at 12 h,24 h,48 h and 72 h after knockdown of lncRNA PVT1(P<0.05),and the results of flow cytometry assay showed that apoptosis level as well as the ROS level of MG-63 cells were significantly increased after knockdown of lncRNA PVT1(P<0.05),and that the results of the combination of the lncRNA PVT1 knockdown group were reversed by miR-106a-5p inhibitor relative to the lncRNA PVT1 knockdown group(P<0.05).miR-106a-5p inhibitor reversed the results in the lncRNA PVT1 knockdown group(P<0.05).RT-qPCR assay showed that miR-106a-5p gene expression increased and MDM4 gene expression was inhibited after knockdown of lncRNA PVT1(P<0.05),and Western blot showed that lncRNA PVT1 inhibited MDM4 gene expression(P<0.05),and lncRNA PVT1 inhibited MDM4 gene expression(P<0.05).Western blot results showed that knockdown of lncRNA inhibited MDM4,HO1,GPX4 and FTH protein levels after PVT1(P<0.05).Conclusion In human osteosarcoma MG-63 cells,inhibition of lncRNA PVT1 expression can promote the expression of miR-106a-5p,facilitate ferroptosis and apoptosis,and inhibit proliferation.

关 键 词：骨肉瘤 lncRNA PVT1 miR-106a-5p 铁死亡 增殖 凋亡 

分 类 号：R738.1[医药卫生—肿瘤]