咪康唑抑制肺癌细胞外泌体分泌机制与生物学效应探讨

Mechanism and biological effects of miconazole on inhibiting exosome secretion in lung cancer cells

目的探讨咪康唑调控肺癌细胞外泌体分泌的机制,及其抑制肿瘤效应。方法以人肺癌细胞A549、H460和人正常肺上皮细胞BEAS-2B为模型,采用电镜观察和外泌体定量试剂盒对外泌体进行定性和定量分析。实时荧光定量PCR和蛋白质印迹法检测EBP蛋白在不同细胞中的表达以及咪康唑和siRNA的靶向效应;CCK-8法检测A549细胞的增殖活性。结果肺癌细胞A549外泌体数量为120×10^(7) mL^(-1),H460细胞的外泌体数量为100×10^(7) mL^(-1),都大于正常肺上皮细胞的外泌体数量(72×10^(7) mL^(-1)),差异有统计学意义(t=13.460,P=0.001;t=19.210,P=0.009)。采用不同浓度咪康唑处理后,能够减少两种肺癌细胞的外泌体分泌数量,并且呈现典型的浓度依赖性,5、10和20μmol/L咪康唑处理后A549细胞分泌的外泌体数量分别为(127.77±7.72)×10^(7) mL^(-1)、(105.38±2.33)×10^(7) mL^(-1)和(72.26±14.48)×10^(7) mL^(-1),均少于对照组(0μmol/L咪康唑)的(139.79±7.70)×10^(7) mL^(-1),差异有统计学意义,P值分别为0.049、0.005和<0.001;H460细胞的外泌体数量分别为(95.83±6.31)×10^(7) mL^(-1)、(79.63±8.65)×10^(7) mL^(-1)和(63.91±11.05)×10^(7) mL^(-1),均少于对照组的(109.29±6.38)×10^(7) mL^(-1),差异具有统计学意义,P值分别为0.046、0.006和<0.001。咪康唑能够降低肺癌细胞EBP蛋白相对含量,而不影响CYP51和TM7SF2。CCK8细胞增殖实验结果显示,4d后外泌体组的光密度值为1 103.00±137.15,明显高于无外泌体对照组的751.67±32.93(t=4.374,P<0.001),而咪康唑处理后的外泌体组为885.33±61.01,也低于外泌体组,t=2.853,P=0.049。结论咪康唑能够靶向肺癌细胞中的EBP蛋白,通过降低EBP稳定性参与调控肺癌细胞的外泌体分泌和旁分泌作用,抑制肺癌的进展。

Objective To explore whether miconazole can regulate the secretion of exosomes of lung cancer cells and produce tumor inhibition effect.Methods Human lung cancer cells A549,H460 and human normal lung epithelial cells BEAS-2 Bwere used as models for qualitative and quantitative analysis of exosomes by electron microscopy and exosome quantitative kit.Real-time quantitative PCR and Western blotting were used to detect the expression of EBP in different cells,the targeting effect of miconazole and siRNA.The proliferation activity of A549 cells was detected by CCK-8 method.Results The number of exosomes from lung cancer cells A549 and H460 were 120×10^(7) ml^(-1) and 100×10^(7) ml^(-1) respectively,which were significantly higher than that from normal lung epithelial cells(72×10^(7) ml^(-1)),t=13.460,P=0.001;t=19.210,P=0.009,respectively.Treatment with different concentrations of miconazole reduced the amount of exosomes in both lung cancer cells,and showed a typical concentration-dependent,when A549 and H460 cells were treated with 5,10,20μmol/L miconazole,the number of exosomes secreted by A549 cells were(127.77±7.72)×10^(7) ml^(-1),(105.38±2.33)×10^(7) ml^(-1),(72.26±14.48)×10^(7) ml^(-1),respectively,and the number of exosomes in control group(0μmol/L miconazole)was(139.79±7.70)×10^(7) ml^(-1).The exosomes number in A549 cells at the 5,10,20μmol/L miconazole groups were less than that in the control group,Pvalues were 0.049,0.005 and<0.001,respectively.The number of exosomes secreted by H460 cells at 5,10,20μmol/L miconazole treatment concentrations were(95.83±6.31)×10^(7) ml^(-1),(79.63±8.65)×10^(7) ml^(-1),(63.91±11.05)×10^(7) ml^(-1),respectively,and the number of exosomes in control group(0μmol/L miconazole)was(109.29±6.38)×10^(7) ml^(-1).The exosomes number in A549 cells at the 5,10,20μmol/L miconazole groups were less than that in the control group,the difference was statistically significant,Pvalues were 0.046,0.006 and<0.001,respectively.Miconazole could reduce the relative content of EBP protein in lung cancer cells without affecting CYP51,TM7 SF2.The results of CCK8 cell proliferation test showed that the absorbance of the exosome group was 1103.00±137.15 after 4 days,which was significantly higher than that of the exosome control group(751.67±32.93),t=4.374,P<0.001.The absorbance of the exosome group after miconazole treatment was 885.33±61.01,lower than the exosomes group,t=2.853,P=0.049.Conclusion Miconazole can target EBP protein in lung cancer cells and participate in regulating the secretion and paracrine of exosomes in lung cancer cells by reducing EBP stability,and thus inhibit the progression of lung cancer.