目的 探讨膜相关蛋白NUMB调控肿瘤增殖的机制。方法 构建NUMB基因敲除载体,转染Hela细胞后，经抗性筛选获得稳定细胞株，通过实时定量PCR（qRT-PCR）和Western blot检测NUMB基因和蛋白表达,验证NUMB基因敲除效果。采用裸鼠成瘤实验研究NUMB基因敲除对肿瘤增殖的影响；利用流式细胞术和Brdu标记实验检测NUMB缺失对细胞周期的影响；在体外模型中检测细胞周期各个时期多种调控蛋白的表达水平。结果 Western blot及qRT-PCR结果表明NUMB基因敲除的体外模型构建成功；裸鼠成瘤实验显示NUMB基因敲除促进肿瘤增殖；流式细胞术和Brdu标记实验表明NUMB基因缺失加速细胞周期G1/S期转换，促进肿瘤细胞增殖；Western blot检测发现NUMB基因敲除组细胞周期调控蛋白Cyclin-E的表达增加，而P27的表达降低。 结论 NUMB通过调控Cyclin-E以及P27的蛋白水平影响细胞周期，进而调控肿瘤增殖。
英文摘要:
Objective To study the specific mechanism of NUMB regulate tumor proliferation. Methods A stable cell line by knocking down NUMB in Hela was eatablished and the Western blot and qRT-PCR was applied to confirm the knocking out of NUMB. The effect on tumor proliferation in the absence of NUMB was investigated by observing tumor growth in nude mice.The effect on the cell cycle in the absence of NUMB was detected by flow cytometry and Brdu assay. Finally, differential expression profiles of various cell cycle regulatory proteins was detected by using Western blot analysis. Results Western blot and qRT-PCR results indicated that NUMB was specifically and efficiently knocked down in the Hela stable cell line. Tumor growth experiments demonstrated that NUMB depletion significantly promoted the tumor proliferation. Flow cytometry and Brdu assay indicated that NUMB depletion significantly promoted the G1/S transition and enhanced the cell proliferation. Western blot results demonstrated that Cyclin-E protein level was increased in NUMB depletion cell, whereas expression of P27 protein was decreased. Conclusion NUMB might be involved in cell cycle process by regulating Cyclin-E and P27 protein level and thereby has an effect on tumor proliferation.
