微小RNA-628-3p调控的DNA解螺旋酶抑制骨肉瘤细胞的增殖  

MicroRNA-628-3p regulated DNA helicase Bloom’s syndrome helicase inhibits the proliferation of osteosarcoma cells

作　　者：李毅[1] 刘宏建[2] 许建中[2] 李广恒 朱宇[1] 卢文龙 Li Yi;Liu Hongjian;Xu Jianzhong;Li Guangheng;Zhu Yu;Lu Wenlong(Department of Orthopedics,the Second Affiliated Hospital of Zhengzhou University,Zhengzhou 450014,China;Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Orthopedics,Shenzhen People’s Hospital,Shenzhen 518020,China)

机构地区：[1]郑州大学第二附属医院骨科,450014 [2]郑州大学第一附属医院骨科,450052 [3]深圳人民医院骨科,518020

出　　处：《中华实验外科杂志》2020年第4期757-760,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(81472136)。

摘　　要：目的探讨微小RNA(miRNA,miR)-628-3p调控的DNA解螺旋酶对骨肉瘤细胞增殖的影响。方法首先收集2013年1月至2017年12月郑州大学第二附属医院骨科收治的股骨骨肉瘤患者60例,同期收集股骨创伤性骨折患者60例作为对照组,采用定量聚合酶链反应(qPCR)检测和比较外周血循环miR-628-3p的表达水平差异,并根据随访结果进行生存分析。以人骨肉瘤细胞U2OS和HOS为研究对象,过表达miR-628-3p,采用细胞计数试剂盒(CCK-8)和集落形成实验检测其增殖活性,采用流式细胞术检测其凋亡率。根据Starbase预测miR-628-3p可与Bloom综合征解螺旋酶(BLM)结合,故进一步采用蛋白质印迹法(Western blot)、qPCR和荧光素酶报告基因实验进行检测。两组间计量资料比较采用独立样本t检验,计数资料比较采用χ2检验。结果骨肉瘤患者外周血和肿瘤组织中miR-628-3p的表达水平(相对表达量分别为0.203±0.047、0.217±0.054)均明显低于正常对照组(相对表达量分别为0.951±0.106、1.013±0.072,P<0.01),差异有统计学意义,受试者工作特征(ROC)曲线分析miR-628-3p在骨肉瘤患者中的表达,曲线下面积(AUC)=0.707(P<0.01),K-M生存分析显示miR-628-3p低表达患者生存时间明显短于高表达患者。CCK-8和集落形成实验检测显示miR-628-3p过表达后可抑制骨肉瘤细胞U2OS和NOS的增殖;流式细胞术检测显示miR-628-3p过表达后可促进骨肉瘤细胞的凋亡;qPCR和Western blot检测显示miR-628-3p过表达后,BLM的表达水平明显低于miR-628-3p nc组(P<0.01),差异有统计学意义。荧光素酶报告基因实验显示miR-628-3p mimc与BLM-mt共转染骨肉瘤细胞U2OS和NOS后,其荧光值明显低于其他转染组(U2OS:0.208±0.017比1.082±0.046,t=4.698,P<0.01;HOS:0.224±0.047比0.966±0.053,t=5.548,P<0.01),差异有统计学意义。裸鼠成瘤证实miR-628-3p过表达后肿瘤大小和重量明显低于miR-628-3p nc组[(1.419±0.274) g比(2.825±0.148) g,t=3.717,P<0.01],差异有Objective To investigate the effect of microRNA(miRNA,miR)-628-3p regulated DNA helicase Bloom’s syndrome helicase(BLM)on the proliferation of osteosarcoma cells.Methods Firstly,60 patients with femur osteosarcoma were collected from January 2013 to December 2017 in our hospital.At the same time,60 patients with traumatic fracture of femur were collected as control group.The expression levels of circulating miR-628-3p in peripheral blood and tissues were detected and compared by qPCR,and survival analysis was carried out according to the follow-up results.Overexpression of miR-628-3p in human osteosarcoma cells U2OS and HOS were studied by cell counting kit-8(CCK-8)and colony formation assays,and apoptotic rate was detected by flow cytometry.Starbase predicted that miR-628-3p could bind to DNA helicase BLM,therefore,Western blotting,quantitative polymerase chain reaction(qPCR)and luciferase reporter assay were furtherly performed.Results The expression of miR-628-3p in peripheral blood and tumor tissue of osteosarcoma patients was significantly lower than that of normal control group(P<0.01).The expression of miR-628-3p in osteosarcoma patients was analyzed by receiver operating characteristic(ROC)curve.Area under curve(AUC)=0.707(P<0.01).The survival time of patients with low expression of miR-628-3p was significantly shorter than that of patients with high expression.CCK-8 and colony formation assay showed that over-expression of miR-628-3p inhibited the proliferation of U2OS and NOS in osteosarcoma cells;flow cytometry showed that over-expression of miR-628-3p promoted the apoptosis of osteosarcoma cells;qPCR and Western blotting showed that over-expression of miR-628-3p resulted in lower expression of BLM than that of miR-628-3p nc group(P<0.01).Luciferase reporter assay showed that the fluorescence values of osteosarcoma cells U2OS and NOS co-transfected with miR-628-3p mimc and BLM-mt were significantly lower than those of other transfection groups(U2OS:0.208±0.017 vs.1.082±0.046,t=4.698,P<0.01;HOS:0.22

关 键 词：骨肉瘤 微小RNA-628-3p DNA解螺旋酶 Bloom综合征解螺旋酶 细胞增殖 

分 类 号：R738.1[医药卫生—肿瘤]