Notch3诱导人小细胞肺癌H446细胞G_0/G_1期阻滞及其机制

目的探究Notch3对人小细胞肺癌H446细胞增殖和细胞周期的影响及其分子机制。方法将人Notch3真核表达质粒、人HES1真核表达质粒、人鼠双微基因2(MDM2)基因沉默表达质粒及其空质粒分别转染H446细胞,采用流式细胞术检测细胞周期,采用RT-PCR法检测Notch3和MDM2的mRNA水平,采用Western blotting方法检测Notch3、HES1、MDM2、Rb和P21蛋白表达水平。结果 Notch3可抑制H446细胞增殖并导致G_0/G_1期阻滞(P0.05),上调HES1蛋白表达水平和下调MDM2蛋白表达水平(P0.05);HES1高表达也能使MDM2蛋白表达水平下降(P0.05),但对其mRNA水平无影响;沉默MDM2基因可抑制H446细胞增殖(P0.05),并使H446细胞中Rb和P21蛋白表达水平升高(P0.05)。结论 Notch3可通过HES1抑制MDM2蛋白表达,进而上调Rb和P21蛋白表达水平,抑制H446细胞增殖并阻滞H446细胞于G_0/G_1期。

Molecular mechanism of cell cycle arrest induced by Notch3 in human small cell lung cancer H446 cells

Objective To investigate the effect of Notch3 on proliferation and the cell cycle in human small cell lung cancer H446 cells and its possible mechanism. Methods H446 cells were stably transfected with human Notch3 and HES1 expressing plasmid separately. Short hairpin RNA (shRNA) was used to knock down MDM2 in H446 cells. The cell cycle was studied by flow cytometry. The mRNA expression levels of Notch3 and MDM2 were measured by RT-PCR. Western blotting was performed to determine the protein expression levels of Notch3, hairy and enhancer of split 1(HES1), murine double minute 2(MDM2), Rb and P21. Results Notch3 suppressed proliferation and induced cell cycle G0/G1 arrest in H446 cells(P<0.05), and up-regulated HES1 and down-regulated MDM2 at protein levels(P<0.05). High expression of HES1 resulted in a lower protein level of MDM2(P<0.05) without affecting the mRNA expression of MDM2. MDM2 knockdown suppressed proliferation and up-regulated Rb and P21 at protein levels(P<0.05). Conclusion Notch3 may suppress proliferation and induce cell cycle arrest by up-regulating HES1, Rb and P21, and down-regulating MDM2.