Notch3诱导人小细胞肺癌H446细胞G0/G1期阻滞及其机制

刘辉 陈红莲 袁磊*

doi:10.16098/j.issn.0529-1356.2016.06.012

PDF(383 KB)

解剖学报 ›› 2016, Vol. 47 ›› Issue (6) : 791-795. DOI: 10.16098/j.issn.0529-1356.2016.06.012

肿瘤生物学

Notch3诱导人小细胞肺癌H446细胞G0/G1期阻滞及其机制

刘辉陈红莲袁磊*

作者信息 +

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

LIU Hui CHEN Hong-lian YUAN Lei*

Author information +

文章历史 +

摘要

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G0/G1期阻滞（P<0.05），上调HES1蛋白表达水平和下调MDM2蛋白表达水平(P<0.05)；HES1高表达也能使MDM2蛋白表达水平下降（P<0.05），但对其mRNA水平无影响；沉默MDM2基因可抑制H446细胞增殖（P<0.05），并使H446细胞中Rb和P21蛋白表达水平升高（P<0.05）。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G0/G1期。

Abstract

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.

导出引用

刘辉陈红莲袁磊. Notch3诱导人小细胞肺癌H446细胞G0/G1期阻滞及其机制[J]. 解剖学报. 2016, 47(6): 791-795 https://doi.org/10.16098/j.issn.0529-1356.2016.06.012

LIU Hui CHEN Hong-lian YUAN Lei. Molecular mechanism of cell cycle arrest induced by Notch3 in human small cell lung cancer H446 cells[J]. Acta Anatomica Sinica. 2016, 47(6): 791-795 https://doi.org/10.16098/j.issn.0529-1356.2016.06.012

参考文献

［1］Sharp A, Bhosle J, Abdelraouf F, et al. Development of molecularly targeted agents and immunotherapies in small cell lung cancer［J］. Eur J Cancer, 2016, 60:26-39.

［2］Carter BW, Glisson BS, Truong MT, et al. Small cell lung carcinoma: staging, imaging, and treatment considerations［J］. Radiographics, 2014, 34(6):1707-1721.

［3］Alvarado-Luna G, Morales-Espinosa D. Treatment for small cell lung cancer, where are we now? -a review［J］. Transl Lung Cancer Res, 2016, 5(1):26-38.

［4］Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells［J］. J Cancer Res Clin Oncol, 2016, 142(5):981-993.

［5］Zhang JX, Han YP, Bai C, et al. Notch1/3 and p53/p21 are a potential therapeutic target for APS-induced apoptosis in non-small cell lung carcinoma cell lines［J］. Int J Clin Exp Med, 2015, 8(8):12539-12547.

［6］Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells［J］. J Cancer Res Clin Oncol, 2016, 142(5):981-993.

［7］Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis［J］. Sci Rep, 2015, 5:10338.

［8］Shi C, Qian J, Ma M, et al. Notch 3 protein, not its gene polymorphism, is associated with the chemotherapy response and prognosis of advanced NSCLC patients［J］. Cell Physiol Biochem, 2014, 34(3):743-752.

［9］Ou WB, Zhu J, Eilers G, et al. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status［J］. Oncotarget, 2015, 6(12):10510-10520.

［10］Zhu Y, Regunath K, Jacq X, et al. Cisplatin causes cell death via TAB1 regulation of p53/MDM2/MDMX circuitry［J］. Genes Dev, 2013, 27(16):1739-1751.

［11］Yu H, Zou Y, Jiang L, et al. Induction of apoptosis in non-small cell lung cancer by downregulation of MDM2 using pH-responsive PMPC-b-PDPA/siRNA complex nanoparticles［J］. Biomaterials, 2013, 34(11):2738-2747.

［12］Künkele A, De Preter K, Heukamp L, et al. Pharmacological activation of the p53 pathway by nutlin-3 exerts anti-tumoral effects in medulloblastomas［J］. Neuro Oncol, 2012, 14(7):859-869.

［13］Park C, Jeong JS, Jeong JW, et al. Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G1 phase［J］. Asian Pac J Trop Med, 2016, 9(4):344-350.

［14］Hu H, Dong Z, Tan P, et al. Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner［J］. Oncotarget, 2016, 7(3):3171-3185.

［15］Li JP,Cai ShZh, Liu J, et al. Replicative senescence characteristics of human leukemia cell line (K562) induced by ginsenoside Rg1［J］. Acta Anatomica Sinica, 2012, 43(4):473-478.(in Chinese)

李建平, 蔡世忠, 刘俊, 等. 人参皂苷Rg1诱导人白血病K562细胞复制性衰老的机制［J］. 解剖学报, 2012, 43(4):473-478.

［16］Bhattacharya S, Ghosh MK. HAUSP, a novel deubiquitinase for Rb-MDM2 the critical regulator［J］. FEBS J, 2014, 281(13):3061-3078.

［17］Inoue K, Fry EA, Frazier DP. Transcription factors that interact with p53 and Mdm2［J］. Int J Cancer, 2016, 138(7):1577-1585.

［18］Zhou R, Lu Z, Liu K, et al. Platycodin D induces tumor growth arrest by activating FOXO3a expression in prostate cancer in vitro and in vivo［J］. Curr Cancer Drug Targets, 2015, 14(9):860-871.

［19］Giovannini C, Minguzzi M, Baglioni M, et al. Suppression of p53 by Notch3 is mediated by Cyclin G1 and sustained by MDM2 and miR-221 axis in hepatocellular carcinoma［J］. Oncotarget, 2014, 5(21):10607-10620.

［20］Kim D, Song J, Jin EJ. MicroRNA-221 regulates chondrogenic differentiation through promoting proteosomal degradation of slug by targeting Mdm2［J］. J Biol Chem, 2010, 285(35):26900-26907.