Effect of 5-Aza-CdR on Growth and Invasion Ability of Non-small Cell Lung Cancer A549 and Expression of TFPI-2 Gene mRNA in vitro
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摘要: 目的 探讨5-氮杂-2′-脱氧胞苷(5-aza-2-deoxycytidine，5-Aza-CdR)干预对非小细胞肺癌(nonsmall cell non cancer, NSCLC)细胞株A549生长增殖及其组织因子途径抑制物2(tissue factor pathway inhibitor-2，TFPI-2)基因表达的影响，同时探讨5-Aza-CdR能否通过恢复TFPI-2基因表达抑制非小细胞肺癌A549细胞侵袭能力。方法 用不同浓度的5-Aza-CdR处理A549细胞株，MTT法检测药物处理24、48、72 h后的细胞增殖活性，流式细胞仪(fl ow cytometry, FCM)法检测药物处理72 h后细胞周期分布，Real-time PCR技术检测药物处理72h后A549细胞TFPI-2基因mRNA的表达，Transwell小室法测定药物处理24h后A549细胞体外侵袭能力的变化。 结果 MTT检测显示不同浓度5-Aza-CdR处理A549细胞24、48、72h后，细胞的生长受到抑制，且抑制作用呈明显的剂量和时间依赖性。FCM检测分析显示0、1、5、10 μM 5-Aza-CdR处理A549细胞72 h 后，细胞增殖指数逐渐降低，分别为（30.43±0.99)%、（23.89±0.83)%、（16.19±0.34)%、（6.49±0.55%（P＜0.05）。Real-timePCR检测显示0、1、5、10 μM的5-Aza-CdR处理A549细胞72 h后，相对mRNA表达水平分别为（1±0)、（1.49±0.14)、（1.86±0.09)、（5.80±0.15)（P<0.05），TFPI-2 基因mRNA表达呈明显的上升趋势，且随着药物浓度增加而增加。Transwell小室法检测显示每一高倍镜下平均穿膜细胞数分别为（316.15±18.7)、（84.15±12.14)、（28.85±7.13)、（14.35±3.33)，均明显低于对照细胞（P<0.05）。结论 5-Aza-CdR能通过降低TFPI-2基因的甲基化而恢复其在非小细胞肺癌细胞株A549细胞中的表达，并抑制A549细胞的增殖和侵袭。
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关键词:
- 非小细胞肺癌 /
- 5-氮杂-2′-脱氧胞苷 /
- 组织因子途径抑制物2 /
- 甲基化
Abstract: Objective To explore the effect of 5-aza-2-deoxycytidine(5-Aza-CdR) on the proliferation of non-small cell lung cancer cell line A549 and the expression of tissue factor pathway inhibitor 2 (TFPI-2) gene mRNA, and to explore whether 5-Aza-CdR could inhibit the invasion capacity of NSCLC A549 cell via TFPI-2 gene. Methods A549 cells were treated with 5-Aza-CdR at different concentrations. The proliferation of A549 cells was detected by MTT assay after treatment for 24 h, 48 h and 72 h, respectively. Cell cycle of A549 cells were analyzed by fl ow cytometer(FCM) after treatment for 72 h. The expression of TFPI-2 gene mRNA was detected by real time polymerase chain reaction(real-time PCR) after treatment for 72 h, and invasion capacity of A549 cells was detected by Transwell assay after treatment for 24 h. Results MTT showed that A549 cell growth had been restrained after treatment at different concentrations for 24 h, 48 h and 72 h, respectively, in an obvious dose- and time-dependence manner. FCM and real-time PCR showed that after treatment at the concentration of 0, 1, 5, 10 μM for 72 h respectively, proliferation indexes of A549 cells were (30.43±0.99)%, (23.89±0.83)%, (16.19±0.34)%, (6.49±0.55)%(P<0.05), and relative mRNA expression levels of TFPI-2 gene were (1±0), (1.49±0.14), (1.86±0.09), (5.80±0.15)(P<0.05), and TFPI-2 gene mRNA expression had increased along with the increasing concentration of 5-Aza-CdR. Transwell small chamber assay showed that the average numbers of cells passed the membrane was (316.15±18.7), (84.15±12.14), (28.85±7.13) and (14.35±3.33) at the concentration of 0, 1, 5, 10 μM respectively(P<0.05). Conclusion 5-Aza-CdR could restore TFPI-2 gene expression in NSCLC cell line A549 cells and restrain the proliferation and invasion capacity of A549 cells by reducing TFPI-2 gene methylation. -
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