黑色素瘤来源的外泌体miR-23a在黑色素瘤达拉非尼耐药中的机制研究

Mechanistic study of melanoma-derived exosomal miR-23a in dabrafenib resistance in melanoma

目的对黑色素瘤来源的外泌体miR-23a在黑色素瘤达拉非尼耐药中的机制进行分析和研究。方法选择恶性黑色素瘤(MM)细胞系A375,用(0、30、100、300 nM)浓度的达拉非尼对其进行24 h处理,再用miR-23a模拟物转染对300 nM的A375细胞进行转染,利用免疫印迹(Western Blot)和实时荧光定量聚合酶链式反应(qPCR)检测和比较转染前后A375细胞中的miR-23a、LC3Ⅰ/Ⅱ、活性氧(ROS)、caspase-3、肿瘤坏死因子-α(TNF-α)、caspase-8表达水平;检测和比较恶性黑色素瘤细胞内被用不同浓度的达拉非尼处理前后LC3Ⅰ/Ⅱ表达的水平;分析miR-23a与耐药相关通路上各个因子的相关性。结果将A375细胞系用不同浓度的达拉非尼(0、30、100、300 nM)处理,收集细胞。经Western Blot检测LC3Ⅰ/Ⅱ蛋白表达水平,并行qPCR分析,发现LC3Ⅰ/Ⅱ水平随达拉非尼浓度的升高而增加,说明达拉非尼以剂量依赖性方式显著激活了自噬信号。转染后,miR-23a、ROS、caspase-3、TNF-α、caspase-8的表达水平分别为(47.45±13.24)、(59.34±14.46)、(40.56±10.23)、(49.23±15.34)、(50.34±8.78),均高于转染前的(26.16±8.23)、(49.55±13.36)、(25.16±5.12)、(30.49±11.54)、(33.53±13.36),差异具有统计学意义(P<0.05)。Pearson线性相关分析结果显示,miR-23a表达水平与caspase-3、TNF-α、caspase-8、TGF-β、LC3Ⅰ/Ⅱ呈正相关(P<0.05),与ROS无相关性(P>0.05)。结论达拉非尼以剂量依赖性方式可以显著激活自噬信号,达拉非尼耐药恶性黑色素瘤细胞中的miR-23a表达水平上调后,能够促使耐药细胞发生自噬而导致凋亡加速,这可能就是miR-23a在黑色素瘤达拉非尼耐药中发生影响的可能机制。

Objective To analyze and study the mechanism of melanoma-derived exosomal miR-23a in dabrafenib resistance in melanoma.Methods The malignant melanoma(MM)cell line A375 was selected and treated with(0,30,100,300 nM)concentration of darafenib for 24 h,and then transfected with miR-23a mimics on A375 cells at 300 nM.The expression level of miR-23a,LC3Ⅰ/Ⅱ,reactive oxygen species(ROS),caspase-3,tumor necrosis factor-α(TNF-α),caspase-8 in A375 cells before and after transfection were detected by immunoblotting(Western Blot)and real-time fluorescence quantitative polymerase chain reaction(qPCR)and then compared.The expression levels of LC3Ⅰ/Ⅱin malignant melanoma cells before and after treatment with different concentrations of dabrafenib were detected and compared,and the correlation between miR-23a and various factors in drug resistance-related pathways was analyzed.Results A375 cell lines were treated with different concentrations of dalafenib(0,30,100,300 nM)and the cells were collected.The expression level of LC3Ⅰ/Ⅱprotein was detected by Western Blot,and the qPCR analysis showed that the level of LC3Ⅰ/Ⅱincreased with the increase of dabrafenib concentration,indicating that dabrafenib significantly activated autophagy signal in a dose-dependent manner.After transfection,the expression levels of miR-23a,ROS,caspase-3,TNF-αand caspase-8 were(47.45±13.24),(59.34±14.46),(40.56±10.23),(49.23±15.34)and(50.34±8.78),which were higher than(26.16±8.23),(49.55±13.36),(25.16±5.12),(30.49±11.54)and(33.53±13.36)before transfection,and the differences were all statistically significant(P<0.05).Pearson linear correlation analysis showed that the expression level of miR-23a was positively correlated with caspase-3,TNF-α,caspase-8,TGF-β,LC3Ⅰ/Ⅱ(P<0.05),but had no correlation with ROS(P>0.05).Conclusion Dabrafenib can significantly activate autophagic signaling in a dose-dependent manner.Upregulation of expression level of miR-23a in dabrafenib-resistant malignant melanoma cells can promote the autophagy of drug-resistant cells and lead to accelerated apoptosis,which may be the possible mechanism for the effect of miR-23a in the occurrence of dalafenib resistance in melanoma.