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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 < 0.01）。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。 Abstract: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 nonsense 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 plasmid 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, CD133, and nestin in the cells transfected with SNB19-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 (χ 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. -
Key words:
- Glioma /
- MicroRNA /
- Glioma stem cells /
- Differentiation
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图 1 SNB19-miR-218及SNB19-scr亚细胞系EGFP表达情况（荧光显微镜×100）
A：SNB19-miR-218亚细胞系；B：SNB19-scr亚细胞系
Figure 1. The EGFP expression in SNB19-miR218 and SNB19-scr subcell lines
图 2 SNB19-miR-218及SNB19-scr亚细胞系中CD133和Nestin的表达
Figure 2. The expression levels of CD133 and Nestin in SNB19-miR218 and SNB19-scr sub-cell lines
图 3 SNB19-miR-218及SNB19-scr亚细胞系GFAP免疫细胞化学染色结果（ABC法×200）
A：SNB19-miR-218亚细胞系；B：SNB19-scr亚细胞系
Figure 3. Immunocytochemistry of GFAP in SNB19-miR218 and SNB19-scr sub-cell lines
图 4 SNB19细胞系低拷贝转染携带EGFP报告基因的miR-218表达质粒后CD133及Nestin免疫荧光检测结果（CD133及Nestin检测信号为红色荧光，EGFP释放出绿色荧光，DAPI复染的细胞核释放出蓝色荧光）
Figure 4. Immunofluorescence results of CD133 and Nestin in the SNB-19 glioblastoma cell line transfected with low copy numbers of miR-218 expression plasmids
表 1 不同SNB19亚细胞系中CD133和Nestin相对表达水平
Table 1. The comparisons of the CD133 and Nestin expression levels of the SNB-19 derived sub-cell lines
表 2 不同SNB-19亚细胞系GFAP阳性标记指数（LI）的比较x±s
Table 2. The comparison of the GFAP labeling index between SNB-19 derived sub-cell lines
表 3 EGFP（+）或（-）SNB-19细胞亚群CD133及Nestin阳性细胞构成比的比较
Table 3. Comparison of the constituent ratios of CD133- and Nestin-positive cells in EGFP-positive and EGFP-negative subpopulations
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