摘要
目的:观察miR-218对胶质母细胞瘤细胞系成熟分化的影响。方法:通过质粒转染和G418筛选建立稳定过表达miR-218的SNB-19亚细胞系及对照亚细胞系。利用qRT-PCR及Western blot检测稳定细胞系中miR-218、CD133及Nestin表达水平。用GFAP免疫细胞化学染色评价其成熟分化水平,通过免疫荧光观察转染miR-218表达质粒后CD133及Nestin阳性细胞的分布情况。结果:过表达miR-218的亚细胞系中miR-218、CD133及Nestin表达水平分别是对照细胞的26.23倍、17.9%及54.2%,过表达miR-218细胞的GFAP阳性标记指数为(96.0±3.81)%显著高于对照细胞(49.6±5.13)%(t=16.24,P<0.0)1)。免疫荧光显示CD133及Nestin的下调特异性发生于成功转染miR-218表达质粒的细胞。结论:miR-218可通过促进胶质瘤干细胞的成熟分化显著下调CD133及Nestin的表达水平,该作用可能是miR-218抑制胶质瘤细胞增殖的重要机制之一。
Objective： To investigate the stimulatory effects of miR-218 on the differentiation of glioblastoma cells. Methods： The SNB-19 glioblastoma subcell line overexpressing miR-218 （ SNB19-miR218 ） and the control subcell line overexpressing a non- sense scrambled sequence （ SNB19-scr ） were established via plasmid transfection and G418 screening. The expression levels of miR-218, CD133, and nestin were detected via quantitative reverse transcription polymerase chain reaction and western blot analysis, whereas the differentiation status was assessed via glial fibrillary acidic protein （ GFAP ） immunocytochemistry. Using low-copy plas- mid transfected cells, the distribution of CD133- and nestin-positive cells was detected via immunofluorescence against an enhanced green fluorescent protein （ EGFP ） reporter. Results： The expression levels of miR-218, CD 133, and nestin in the cells transfected with SNBI9-miR218 were 26.23-fold, 17.9%, and 54.2% of those of SNB19-scr. The GFAP labeling index of SNB19-miR218 （ 96.0%± 3.81% ） was significantly higher than that of SNB19-scr （ 49.6% ±5.13%, t = 16.24, P 〈 0.0001 ）. Immunofluorescence showed that the constituent ratios of the CD133- and nestin-positive cells in the EGFP （ ＋ ） and （ - ） subpopulations were significantly uneven （ X 2 = 586.38 and 658.53, respectively, P 〈 0.0001 ）, confirming that the CD133 and nestin were downregulated specifically in the tumor cells successfully transfected with the miR-218 expression plasmid. Conclusion： miR-218 promotes glioma stem cell differentiation, and therefore downregulates CD133 and nestin expression. This effect may be an important mechanism for inhibiting the proliferation of glioma cells.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2012年第9期532-535,共4页
Chinese Journal of Clinical Oncology
基金
国家973计划分项目(编号:2010CB529405)
天津市应用基础及前沿技术研究计划重点项目(编号:10JCZDJCl9400)
天津市科技创新体系及条件平台建设计划重大项目(编号:10SYSYJC28800)资助~~
