黑色素瘤SENP1蛋白质参与达卡巴嗪耐药性的探究

Involvement of SENP1 protein in resistance of dacarbazine in melanoma

目的探究与黑色素瘤耐药性相关的基因及信号通路,揭示其与黑色素瘤耐药性的相关性。方法以A375及M14黑色素瘤细胞为研究对象,通过逐渐提高达卡巴嗪(dacarbazine,DTIC)的浓度获得耐药性黑色素瘤细胞株,采用转录物组学研究耐药性黑色素瘤细胞系中显著性变化的基因及通路,利用实时荧光定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)、蛋白质杂交(Western blotting,WB)等对变化的基因进行验证。结果(1)成功构建了黑色素瘤耐药细胞株:通过DTIC小剂量逐步增加的方法成功建立了耐药型的黑色素瘤细胞系A375与M14,通过计算其对DTIC的半抑制浓度值(the half maximal inhibitory concentration,IC50),确定了细胞对DTIC的敏感性发生了显著的变化;通过流式细胞技术检测,发现耐药的黑色素瘤细胞具有显著抗DTIC引发的凋亡的能力。(2)发现了黑色素瘤耐药性相关的基因及信号通路:利用建立的耐DTIC的黑色素瘤细胞系,进行了全基因组转录测序和分析,发现了类泛素特异性蛋白酶1(SUMO-specific protease 1,SENP1)的高表达和蛋白激酶Hippo信号通路的异常活化相关。(3)SENP1异常表达可能参与DTIC耐药:WB检测野生型和耐药型黑色素瘤细胞系发现在耐药的细胞中,SENP1与YAP表达都上调。(4)通过基因敲除证实SENP1参与DTIC耐药,蛋白质相互作用实验初步证实SENP1对YAP存在去泛素化调控作用。结论SENP1与DTIC耐药性存在正相关关系,其异常上调可能导致Hippo信号通路发生变化使得黑色素瘤对DTIC耐受性的提升。

Objective To investigate the genes and signaling pathways associated with drug resistance in melanoma and establish their relationship with melanoma drug resistance.Methods A375 and M14 melanoma cells were used as the experimental model to develop a drug-resistant melanoma cell line by gradually increasing the concentration of dacarbazine(DTIC).Transcriptomics was employed to analyze significant changes in genes and pathways within the drug-resistant melanoma cell line.Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB)were conducted to validate the altered genes.Results(1)Successful construction of melanoma drug-resistant cell lines:drug-resistant cell lines,namely A375 and M14,were successfully established by gradually increasing the dosage of DTIC.The determination of their the half maximal inhibitory concentration(IC50)values to DTIC indicated a significant change in cell sensitivity.Flow cytometry analysis revealed that drug-resistant melanoma cells exhibited a notable ability to resist apoptosis induced by DTIC.(2)Identification of genes and signaling pathways associated with melanoma drug resistance:transcriptomic analysis by the established DTIC-resistant melanoma cell lines demonstrated that increased expression of SENP1 was correlated with abnormal activation of the Hippo signaling pathway.(3)Implication of aberrant expression of ubiquitin-specific protease SENP1 in DTIC resistance:WB analysis of wild-type and drug-resistant melanoma cell lines revealed upregulation of both SENP1 and YAP expressions in drug-resistant cells.(4)The involvement of SENP1 in DTIC resistance was confirmed by gene knockout and the effect of SENP1 on ubiquitination of YAP was preliminarily confirmed by protein interaction experiments.Conclusion A positive correlation existed between SENP1 and DTIC resistance in melanoma,which may contribute to alterations in the Hippo signaling pathway and enhance melanoma tolerance to DTIC.