摘要
目的探讨泛素连接酶LRSAM1在CD166降解中的作用及其对人神经胶质母细胞瘤细胞增殖、侵袭和迁移的影响。方法人神经胶质母细胞瘤U-87 MG细胞,采用免疫共沉淀法检测LRSAM1与CD166是否存在结合;将对数生长期U-87 MG细胞分为pcDNA3.1-LRSAM1组、pcDNA3.1-NC组和空白对照组,pcDNA3.1-LRSAM1组和pcDNA3.1-NC组细胞分别转染pcDNA3.1-LRSAM1质粒载体和pcDNA3.1空质粒载体,空白对照组细胞正常培养,采用实时荧光定量PCR法检测3组细胞LRSAM1 mRNA相对表达量,采用Western blot法检测LRSAM1蛋白相对表达量,采用免疫荧光染色法检测LRSAM1与CD166表达情况,采用细胞克隆形成实验检测细胞集落形成数目,采用Annexin V-FITC/PI染色检测细胞凋亡率,采用细胞划痕试验检测细胞划痕愈合率,采用Transwell小室实验检测侵袭细胞数。结果 U-87 MG细胞中LRSAM1与CD166存在结合。pcDNA3.1-LRSAM1组细胞LRSAM1 mRNA和蛋白相对表达量(3.26±0.28、0.72±0.06)均高于空白对照组(1.00±0.03、0.18±0.01)和pcDNA3.1-NC组(0.99±0.02、0.19±0.02)(P<0.05),转染成功。U-87 MG细胞LRSAM1与CD166共定位,pcDNA3.1-LRSAM1组LRSAM1荧光染色强度增加,CD166荧光染色强度减弱;转染后pcDNA3.1-LRSAM1组细胞集落形成数目[(69.21±7.54)个]、侵袭细胞数[(33.71±4.17)个]均少于空白对照组[(142.38±14.61)、(68.87±9.42)个]和pcDNA3.1-NC组[(152.40±15.92)、(75.54±9.06)个](P<0.05),细胞凋亡率[(17.41±2.03)%]高于空白对照组[(8.07±0.79)%]和pcDNA3.1-NC组[(8.45±0.88)%](P<0.05),细胞划痕愈合率[(30.16±4.10)%]低于空白对照组[(58.22±5.82)%]和pcDNA3.1-NC组[(62.09±7.13)%](P<0.05);空白对照组与pcDNA3.1-NC组各指标比较差异均无统计学意义(P>0.05)。结论过表达LRSAM1可抑制人神经胶质母细胞瘤U-87 MG细胞增殖、侵袭与迁移,促进细胞凋亡,其机制可能与LRSAM1结合CD166有关。
Objective To explore the role of ubiquitin ligase LRSAM1 in the degradation of CD166 and its effects on the proliferation, invasion and migration of human glioblastoma cells. Methods Immunoprecipitation method was used to detect whether LRSAM1 and CD166 were integrated in human glioblastoma U-87 MG cells. The U-87 MG cells in logarithmic growth phase were divided into pcDNA3.1-LRSAM1 group, pcDNA3.1-NC group and blank control group. The cells in pcDNA3.1-LRSAM1 group and pcDNA3.1-NC group were transfected with pcDNA3.1-LRSAM1 plasmid vector and pcDNA3.1 empty plasmid vector, respectively, and the cells in blank control group were cultured normally. Real-time fluorescence quantitative PCR was used to detect the relative expression of LRSAM1 mRNA, Western blot was used to detect the relative expression of LRSAM1 protein, immunofluorescence staining method was used to detect the expressions of LRSAM1 and CD166, cell colony formation experiment was used to detect the number of cell colonies formed, Annexin V-FITC/PI staining was used to detect the cell apoptosis rate, cell scratch test was used to detect the cell scratch healing rate, and Transwell chamber experiment was used to detect the number of invaded cells. Results LRSAM1 was integrated with CD166 in U-87 MG cells. The relative expressions of LRSAM1 mRNA and protein were higher in pcDNA3.1-LRSAM1 group(3.26±0.28, 0.72±0.06) than those in blank control group(1.00±0.03, 0.18±0.01) and pcDNA3.1-NC group(0.99±0.02, 0.19±0.02)(P<0.05), and the transfection was successful. LRSAM1 was co-localized with CD166 in U-87 MG cells. In pcDNA3.1-LRSAM1 group, the fluorescence staining intensity of CD166 decreased and the fluorescence staining intensity of LRSAM1 increased.After transfection,the number of formed cell colonies and the number of invaded cells were less in pcDNA3.1-LRSAM1 group(69.21±7.54,33.71±4.17)than those in blank control group(142.38±14.61,68.87±9.42)and pcDNA3.1-NC group(152.40±15.92,75.54±9.06)(P<0.05),the apoptosis rate was higher in pcDNA3.
作者
梁博
王新军
王建业
周少龙
杨卓
刘荣俊
LIANG Bo;WANG Xin-jun;WANG Jian-ye;ZHOU Shao-long;YANG Zhuo;LIU Rong-jun(Department of Neurosurgery,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450052,China)
出处
《中华实用诊断与治疗杂志》
2021年第5期456-460,共5页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家自然科学基金(81972361)。
