摘要
目的探讨miR-let-7c-5p对膀胱癌细胞恶性行为、血管管腔形成及高迁移率族蛋白(HMGB)2的调控作用。方法将人膀胱癌UM-UC-3细胞分别进行常规培养(空白组)、转染mimics-NC(mimics-NC组)、转染miR-let-7c-5p-mimics(miR-let-7c-5p-mimics组)、转染inhibitor-NC(inhibitor-NC组)及转染miR-let-7c-5p-inhibitor(miR-let-7c-5p-inhibitor组)。采用实时荧光定量聚合酶链反应检测各组细胞miR-let-7c-5p转染成功率;采用Transwell法检测各组细胞侵袭能力;采用划痕试验检测各组细胞迁移能力;采用Matrigel胶小管形成试验检测各组细胞血管生成;采用免疫印迹法检测各组细胞HMGB2、血管内皮生长因子(VEGF)及葡萄糖转运蛋白-1(GLUT-1)蛋白表达水平;采用荧光素酶试验验证miR-let-7c-5p对HMGB2的调控作用。结果空白组、mimics-NC组及inhibitor-NC组miR-let-7c-5p mRNA表达水平、侵袭数目、划痕愈合率、血管管腔数目,HMGB2、VEGF及GLUT-1蛋白表达水平比较,差异均无统计学意义(P>0.05);与空白组、mimics-NC组及inhibitor-NC组比较,miR-let-7c-5p-mimics组miR-let-7c-5p mRNA表达水平升高,侵袭数目、划痕愈合率、血管管腔数目,HMGB2、VEGF及GLUT-1蛋白表达水平均降低,miR-let-7c-5p-inhibitor组miR-let-7c-5p mRNA表达水平降低,侵袭数目、划痕愈合率、血管管腔数目,HMGB2、VEGF及GLUT-1蛋白表达水平均升高,差异均有统计学意义(P<0.05)。荧光素酶试验结果显示,与mimics-NC组比较,miR-let-7c-5p-mimics组HMGB2-Wt水平降低,差异有统计学意义(P<0.05)。结论上调miR-let-7c-5p可明显抑制膀胱癌细胞侵袭、转移及血管生成,其机制与抑制HMGB2蛋白表达相关。
Objective To investigate the effects of miR-let-7c-5p on the malignant behavior,vascular lumen formation and high mobility group protein(HMGB)2 in bladder cancer cells.Methods Human bladder cancer UM-UC-3 cells were divided into routine culture(blank group),transfected with mimics-NC(mimics-NC group),transfected with miR-let-7c-5p-mimics(miR-let-7c-5p-mimics group),transfected with inhibitor-NC(inhibitor-NC group)and transfected with miR-let-7c-5p-inhibitor(miR-let-7c-5p-inhibitor group).Real-time fluorescent quantitative polymerase chain reaction was used to detect the transfection success rate of miR-let-7c-5p in each group.Transwell assay was used to detect cell invasion ability of each group.Migration ability of each group was detected by scratch test.Angiogenesis of cells in each group was detected by Matrigel tube formation test.The protein expression levels of HMGB2,vascular endothelial growth factor(VEGF)and glucose transporter-1(GLUT-1)were detected by Western blot.Luciferase assay was used to verify the regulatory effect of miR-let-7c-5p on HMGB2.Results There was no significant difference in the expression level of miR-let-7c-5p mRNA,invasion number,scratch healing rate,number of vascular lumen,and protein levels of HMGB2,VEGF,and GLUT-1 among the blank group,mimics-NC group,and inhibitor-NC group(P>0.05).Compared with the blank group,mimics-NC group and inhibitor-NC group,the miR-let-7c-5p mRNA expression level in the miR-let-7c-5p-mimics group was increased,the invasion number,scratch healing rate,the number of vascular lumen,and the protein expression levels of HMGB2,VEGF and GLUT-1 were decreased.In the miR-let-7c-5P-inhibitor group,the miR-let-7c-5p mRNA expression level was significantly decreased,while the invasion number,scratch healing rate,the number of vascular lumen,and the protein expression levels of HMGB2,VEGF,and GLUT-1 were significantly increased(P<0.05).Luciferase assay showed that the level of HMGB2-Wt in the miR-let-7c-5p-mimics group was significantly lower than that in the mimics
作者
刘辉勇
董铮
徐涛
柳琦
LIU Huiyong;DONG Zheng;XU Tao;LIU Qi(Department of Urology,Huanggang Central Hospital,Huanggang,Hubei 438000,China)
出处
《检验医学与临床》
CAS
2024年第8期1035-1040,共6页
Laboratory Medicine and Clinic
基金
国家自然科学基金项目(81602221)
湖北省自然科学基金项目(2022CFB799)。
关键词
膀胱癌
血管管腔
高迁移率族蛋白2
侵袭
转移
荧光素酶试验
bladder cancer
vascular lumen
high mobility group box 2
invasion
metastasis
luciferase assay
