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目的 :明确微RNA(microRNA,miRNA)miR-200b是否能通过靶向调控CD133的表达而抑制胶质瘤细胞生长和侵袭的能力,从而揭示miR-200b在抑制肿瘤生长中的分子机制。方法 :首先构建CD133 mRNA3’-非翻译区(3’-untranslated region,3’-UTR)荧光素酶报告载体,通过荧光素酶报告系统观察miR-200b对CD133 mRNA 3’-UTR荧光素酶活性的影响;随后采用脂质体法将miR-200b模拟物(mimics)转入胶质瘤U251细胞中,同时以转入CD133-小干扰RNA(small interfering RNA,siRNA)为阳性对照;采用蛋白质印迹法检测CD133蛋白表达水平的改变;MTS法检测miR-200b mimics对U251细胞增殖活性的影响,Transwell小室法检测对细胞袭侵袭能力的影响;最后,通过干细胞成球实验检测miR-200b mimics对胶质瘤U251干细胞微球体形成的影响。结果 :双荧光素酶报告检测结果显示,miR-200b能特异性地与CD133 mRNA 3’-UTR结合,抑制其荧光素酶的活性。过表达miR-200b的U251细胞中CD133蛋白表达水平明显降低,可显著抑制U251细胞的侵袭能力,而过表达CD133可拮抗miR-200b对U251细胞增殖和侵袭的影响;干细胞成球实验证实,miR-200b能抑制胶质瘤U251干细胞的微球体形成能力。结论 :miR-200b通过靶向调控CD133的表达而抑制胶质瘤细胞增殖、侵袭及干细胞的形成。
OBJECTIVE: To determine whether miR-200b can inhibit the growth and invasion of glioma cells by regulating the expression of CD133, so as to reveal the molecular mechanism of miR-200b in inhibiting tumor growth. Methods: The CD133 mRNA 3'-untranslated region (3'-UTR) luciferase reporter vector was constructed. The luciferase reporter system was used to detect the luciferase activity of miR-200b on 3'-UTR of CD133 mRNA Subsequently, miR-200b mimics were transfected into glioma U251 cells by lipofectamine and transfected into CD133-small interfering RNA (siRNA) as positive control. Western blot The effect of miR-200b mimics on the proliferation activity of U251 cells was detected by MTS assay. The influence of miR-200b mimics on the invasion ability of cells was detected by Transwell chamber assay. Finally, Effect of Stem Cell U251 Stem Cell Microsphere Formation. Results: The results of dual luciferase reporter assay showed that miR-200b could specifically bind to CD133 mRNA 3'-UTR and inhibit its luciferase activity. The expression of CD133 protein in U251 cells overexpressing miR-200b was significantly lower than that in U251 cells, and significantly inhibited the invasion ability of U251 cells. Overexpression of CD133 antagonized the effect of miR-200b on the proliferation and invasion of U251 cells. -200b inhibits the ability of glioma U251 stem cells to form microspheres. Conclusion: miR-200b inhibits the proliferation, invasion and stem cell formation of glioma cells by targeting the expression of CD133.