国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (5): 321-326.doi: 10.3760/cma.j.issn.1673422X.2017.05.001

• 论著 •    下一篇

多聚肌苷酸-多聚胞苷酸诱导肺腺癌A549细胞凋亡的机制

易亮,孙丹,韩倩,柴晓宇,刘新民   

  1. 100000 北京大学第一医院老年科
  • 出版日期:2017-05-08 发布日期:2017-04-19
  • 通讯作者: 刘新民，Email: lxm2128@163.com E-mail:lxm2128@163.com
  • 基金资助:

    北京市自然科学基金（7151011）

Mechanism of Poly(I:C)induced apoptosis in lung adenocarcinoma A549 cells

Yi Liang, Sun Dan, Han Qian, Chai Xiaoyu, Liu Xinmin   

  1. Department of Geriatric, Peking University First Hospital, Beijing 100000, China
  • Online:2017-05-08 Published:2017-04-19
  • Contact: Liu Xinmin E-mail:lxm2128@163.com
  • Supported by:

    Beijing Municipal Natural Science Foundation of China (7151011)

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摘要： 目的研究多聚肌苷酸多聚胞苷酸［Poly(I:C)］对肺腺癌A549细胞活力的影响，探讨Poly(I:C)诱导A549细胞凋亡机制。方法用Poly(I:C)和脂质体3000转染A549细胞，采用四甲基偶氮唑蓝（MTT）检测细胞存活力；采用流式细胞术检测细胞凋亡；Western blotting用于检测凋亡相关蛋白、实时定量PCR检测细胞因子β干扰素（IFNβ）、趋化因子10（CXCL10）的表达。使用pancaspase抑制剂ZVADFMK和caspase8特异性抑制剂ZIETDFMK抑制凋亡蛋白后，观察Poly(I:C)诱导A549细胞凋亡的变化。RNA干扰实验敲低黑色素瘤分化相关抗原5（MDA5）、维甲酸诱导基因Ⅰ（RIGⅠ）的表达，进行上述指标的检测。结果200 ng/ml Poly(I:C)转染后，肺腺癌A549细胞的存活率为74.92%±6.24%，较转染前（95.32%±3.05%）降低（t=2.883，P=0.041）。100、200、400 ng/ml的Poly(I:C)诱导A549细胞凋亡率分别为9.97%±0.88%、23.63%±1.41%、32.57%±2.39%，与对照组(0.74%±0.15%)相比，差异均有统计学意义(t=4.489，P=0.002；t=11.616，P=0.000；t=16.932，P=0.000)。此外，死亡受体途径蛋白如TNF相关的凋亡诱导配体（TRAIL）、cleavedcaspase8、cleavedcaspase3明显升高，同时MDA5/RIGⅠ通路激活以及IFNβ、CXCL10的表达上调。pancaspase抑制剂ZVADFMK、caspase8抑制剂ZIETDFMK使细胞凋亡率分别降至3.17%±0.66%、5.35%±0.64%，与不加抑制剂（15.87%±0.93%）相比，差异有统计学意义（t=8.643，P=0.001；t=6.824，P=0.002）。RIGⅠ、MDA5敲低后，Poly(I:C)诱导的TRAIL、IFNβ、CXCL10表达下调。结论Poly(I:C)能降低A549细胞的存活率，通过激活死亡受体途径诱导A549细胞的凋亡，并且MDA5/RIGⅠ可能参与了该凋亡途径，此过程可能与IFNβ、CXCL10有关。

关键词: 肺肿瘤, 细胞凋亡, 多聚肌苷酸-多聚胞苷酸

Abstract: ObjectiveTo study the effects of Poly(I:C) on lung adenocarcinoma A549 cells viability and illuminate the mechanism of Poly(I:C)induced apoptosis in A549 cells. MethodsA549 cells were transfected with the complex of Poly(I:C) and lipofectamine 3000. The viability of A549 cells was tested by methyl thiazolyl tetrazolium (MTT) method. The apoptotic cells were tested by flow cytometry. The caspase proteins were tested by Western blotting and the expressions of interferonβ (IFNβ) and CXCL10 were assayed by realtime PCR. After employing the pancaspase inhibitor ZVADFMK and caspase8 inhibitor ZIETDFMK, the variation of Poly(I:C) proapoptosis in A549 cells was observed. RNA interfering experiments were employed to knock down melanoma differentiation related antigen 5 (MDA5) or retinoic acidinduced gene Ⅰ (RIGⅠ), and the above indexes were tested. ResultsThe viability of A549 cells was significantly reduced to 74.92%±6.24% after 200 ng/ml Poly(I:C) transfection compared with that before transfection (95.32%±3.05%, t=2.883, P=0.041). The apoptotic rates induced by 100, 200, 400 ng/ml Poly(I:C) were 9.97%±0.88%, 23.63%±1.41%, 32.57%±2.39%, respectively. All of them were higher than that in the control group (0.74%±0.15%), with significant differences (t=4.489, P=0.002; t=11.616, P=0.000; t=16.932, P=0.000). Besides, the death receptor pathway proteins such as TNFrelated apoptosis inducing ligand (TRAIL), cleavedcaspase8 and cleavedcaspase3 increased obviously. MDA5/RIGⅠpathway was also activated dramatically and the expressions of IFNβ, CXCL10 were significantly upregulated. The apoptotic rates reduced to 3.17%±0.66%, 5.35%±0.64% with pancaspase inhibitor ZVADFMK and caspase8 inhibitor ZIETDFMK pretreatment, compared with the control group (15.87%±0.93%), and the differences were statistically significant (t=8.643, P=0.001; t=6.824, P=0.002). Moreover, the expressions of TRAIL, IFNβ and CXCL10 induced by Poly(I:C) were inhibited with MDA5 or RIGⅠ depletion. ConclusionPoly(I:C) can reduce the survival rate of A549 cells and promote the apoptosis mainly by activating the deathreceptor pathway mediated by MDA5/RIGⅠprobably, which may involve in IFNβ, CXCL10

Key words: Lung neoplasms, Apoptosis, Poly(I:C)