国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (2): 65-71.doi: 10.3760/cma.j.issn.1673422X.2019.02.001

• 论著 •    下一篇

靶向沉默Notch1基因对人非小细胞肺癌干细胞增殖和凋亡的影响

 刘恒铫1, 2, 牟艳玲2, 王燕2, 王福文2, 赵国丽1, 3, 王兆朋3, 周淑萍3, 蔡海波4, 张月英3   

  1. 1济南大学 山东省医学科学院医学与生命科学学院，济南250200； 2山东省医学科学院药物研究所 国家卫生和计划生育委员会生物技术药物重点实验室 山东省罕少见病重点实验室，济南250062；3山东省医学科学院基础医学研究所病理学与病理生理学研究室，济南250062；4济宁市第一人民医院胸外科272011
  • 出版日期:2019-02-08 发布日期:2019-04-03
  • 通讯作者: 张月英，Email： zhangyueying828@126.com；王福文，Email： wangfuwww@126.com E-mail:zhangyueying828@126.com; wangfuwww@126.com
  • 基金资助:

    国家自然科学基金（81403150）；山东省自然科学基金（ZR2014HL064）；山东省中医药科技发展计划（2015325）；山东省医学科学院医药卫生科技创新工程

Effect of targeted silencing Notch1 on proliferation and apoptosis of human nonsmall cell lung cancer stem cells 

Liu Hengyao1,2, Mu Yanling2, Wang Yan2, Wang Fuwen2, Zhao Guoli1,3, Wang Zhaopeng3, Zhou Shuping3, Cai Haibo4, Zhang Yueying3   

  1. 1School of Medicine and Life Sciences, University of JinanShandong Academy of Medical Sciences, Jinan 250200, China; 2Institute of Materia Medica, Shandong Academy of Medical Sciences, Key Laboratory for BiotechDrugs of National Health and Family Planning Commission, Key Laboratory for Rare ＆ Uncommon Diseases of Shandong Province, Jinan 250062, China; 3Laboratory of Pathology and Pathophysiology, Institute of Basic Medical Sciences, Shandong Academy of Medical Sciences, Jinan 250062, China; 4Department of Thoracic Surgery, Jining No.1 People′s Hospital, Jining 272011, China
  • Online:2019-02-08 Published:2019-04-03
  • Contact: Zhang Yueying, Email: zhangyueying828@126.com; Wang Fuwen, Email: wangfuwww@126.com E-mail:zhangyueying828@126.com; wangfuwww@126.com

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摘要： 目的  研究靶向沉默Notch1基因对人非小细胞肺癌干细胞增殖和凋亡的影响。方法  选取人肺癌A549细胞和SPC-A-1细胞，分为空白对照组、Nc-shRNA组和Notch1-shRNA组，Nc-shRNA组为阴性对照RNAi慢病毒组，Notch1-shRNA组为Notch1抑制RNAi慢病毒组。采用慢病毒介导的shRNA干扰技术靶向沉默Notch1基因，实时定量聚合酶链反应（qRT-PCR）和Western blotting验证Notch1基因的沉默效果；采用四甲基偶氮唑蓝(MTT)法和成球实验检测细胞增殖能力；采用Annexin V/7-AAD 双染法检测细胞凋亡情况；采用Western blotting检测增殖细胞核抗原（PCNA）、B淋巴细胞瘤-2（Bcl-2）以及Notch1下游基因Hes1的表达情况。结果  qRT-PCR结果显示A549细胞和SPC-A-1细胞中空白对照组、Nc-shRNA组和Notch1shRNA组Notch1相对表达量分别为1.000±0.000、0.937±0.025、0.490±0.036和1.000±0.000、1.077±0.070、0.373±0.038，差异均具有统计学意义（F=359.707，P＜0.001；F=210.455，P＜0.001），进一步两两比较，两种细胞中Notch1-shRNA组Notch1相对表达量明显低于Nc-shRNA组（均P＜0.05）；Western blotting检测A549细胞和SPC-A-1细胞中Notch1蛋白表达的结果与mRNA结果一致。MTT实验显示A549细胞球空白对照组、Nc-shRNA组和Notch1-shRNA组在24 hA值分别为0.209±0.005、0.219±0.009、0.159±0.006，48 h A值分别为0.293±0.004、0.302±0.004、0.205±0.005，72 h A值分别为0.450±0.003、0.430±0.012、0.348±0.017，差异均具有统计学意义（F=79.487，P＜0.001；F=508.664，P＜0.001；F=57.156，P＜0.001），进一步两两比较，24、48、72 h Notch1-shRNA组增殖能力明显低于Nc-shRNA组（均P＜0.05）；SPC-A-1细胞球空白对照组、Nc-shRNA组和Notch1-shRNA组在48 h A值为0.438±0.022、0.412±0.015、0.364±0.010，72 h A值为0.540±0.016、0.519±0.009、0.438±0.019，差异均具有统计学意义（F=15.667，P=0.004；F=37.299，P＜0.001），进一步两两比较，48、72 h Notch1-shRNA组增殖能力明显低于Nc-shRNA组（均P＜0.05）。空白对照组、Nc-shRNA组和Notch1-shRNA组A549细胞成球直径分别为（149.667±6.506）μm、（136.667±7.095）μm、（86.676±7.638）μm，差异具有统计学意义（F=65.940，P＜0.001）；SPC-A-1细胞成球直径分别为（118.667±6.658）μm、（128.000±7.000）μm、（60.675±4.509）μm，差异具有统计学意义（F=105.372，P＜0.001）；进一步两两比较，两种细胞Notch1-shRNA组成球大小明显小于Nc-shRNA组，差异均具有统计学意义（均P＜0.05）。流式细胞术检测A549细胞和SPC-A-1细胞空白对照组、Nc-shRNA组、Notch1-shRNA组凋亡率分别为（0.489±0.014）%、（0.633±0.021）%、（1.683±0.221）%和（1.323±0.194）%、（1.690±0.188）%、（3.017±0.356）%，差异均具有统计学意义（F=77.660，P＜0.001；F=32.200，P=0.001），进一步两两比较，两种细胞中Notch1-shRNA组凋亡率明显高于Nc-shRNA组（均P＜0.05）。Western blotting显示A549细胞空白对照组、Nc-shRNA组和Notch1shRNA组3组间PCNA、Bcl-2、Hes-1表达差异均具有统计学意义（F=155.343，P＜0.001；F=22.576，P=0.002；F=70.108，P＜0.001 ），SPC-A-1细胞空白对照组、Nc-shRNA组和Notch1-shRNA组3组间PCNA、Bcl-2、Hes-1表达差异均具有统计学意义（F=49.419，P＜0.001；F=28.090，P=0.001；F=12.040，P=0.007），进一步两两比较，两种细胞PCNA、Bcl-2、Hes-1表达在Notch1-shRNA组明显低于Nc-shRNA组，差异均具有统计学意义（均P＜0.05）。结论  靶向沉默Notch1能降低肺癌干细胞增殖活性，促进其凋亡，可能与其下游基因Hes-1下调有关。

关键词: 受体,Notch1, 肿瘤干细胞, 癌, 非小细胞肺, 细胞增殖, 细胞凋亡

Abstract: Objective  To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells. Methods  Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group, Nc-shRNA group and Notch1-shRNA group. The Nc-shRNA group was a negative control RNAi lentivirus group, and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group. The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1. The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay. Apoptosis was detected by Annexin V/7-AAD double staining. Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1. Results  The results of qRT-PCR showed that the relative expression levels of Notch1 in control group, Nc-shRNA group and Notch1shRNA group in A549 cells and SPC-A-1 cells were 1.000±0.000, 0.937±0.025, 0.490±0.036 and 1.000±0.000, 1.077±0.070, 0.373±0.038, with statistically significant differences (F=359.707, P<0.001; F=210.455, P<0.001), further paired comparison, the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P<0.05). Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results. MTT assay showed that the 24 h A values of A549 cells in control group, Nc-shRNA group and Notch1shRNA group were 0.209±0.005, 0.219±0.009, 0.159±0.006, 48 h A values were 0.293±0.004, 0.302±0.004, 0.205±0.005, 72 h A values were 0.450±0.003, 0.430±0.012, 0.348±0.017, with statistically significant differences (F=79.487, P<0.001; F=508.664, P<0.001; F=57.156, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of NcshRNA group at 24, 48, 72 h (all P<0.05). The 48 h A values of SPC-A-1 cells in control group, Nc-shRNA group and Notch1shRNA group were 0.438±0.022, 0.412±0.015, 0.364±0.010, 72 h A values were 0.540±0.016, 0.519±0.009, 0.438±0.019, with statistically significant differences (F=15.667, P=0.004; F=37.299, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P<0.05). The sphere sizes of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667±6.506)μm, (136.667±7.095)μm, (86.676±7.638)μm, with statistically significant difference (F=65.940, P<0.001). The sphere sizes of the three groups in SPC-A-1 cells were (118.667±6.658)μm, (128.000±7.000)μm, (60.675±4.509)μm, with statistically significant difference (F=105.372, P<0.001). Further paired comparison, the sphere size of Notch1-shRNA group was significantly smaller than that of Nc-shRNA group in the two kinds of cells (all P<0.05). The apoptosis rates of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were (0.489±0.014)%, (0.633±0.021)%, (1.683±0.221)% and (1.323±0.194)%, (1.690±0.188)%, (3.017±0.356)%, with statistically significant differences (F=77.660, P<0.001; F=32.200, P=0.001), further paired comparison, the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P<0.05). Western blotting showed that the expressions of PCNA, Bcl-2 and Hes-1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F=155.343, P<0.001; F=22.576, P=0.002; F=70.108, P<0.001), and the expressions of PCNA, Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F=49.419, P<0.001; F=28.090, P=0.001; F=12.040, P=0.007). Further paired comparison, the expressions of PCNA, Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells, and the differences were statistically significant (all P<0.05). Conclusion  Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis, which may be related to the down-regulation of its downstream gene Hes1