去泛素化酶JOSD2通过调控DNA损伤修复影响非小细胞肺癌细胞对抗肿瘤药物的敏感性

Deubiquitinating enzyme JOSD2 affects susceptibility of non-small cell lung carcinoma cells to anti-cancer drugs through DNA damage repair

目的:研究去泛素化酶含约瑟芬结构域2蛋白(JOSD2)对非小细胞肺癌(NSCLC)恶性进展的调控作用及其分子机制。方法:从基因表达数据库下载NSCLC转录组表达数据及临床资料,利用主成分分析、limma差异分析和基因表达量分析考察NSCLC中表达水平显著上调的去泛素化酶相关基因;利用KaplanMeier生存分析算法考察不同去泛素化酶对NSCLC患者总生存期的影响;利用基因本体论富集分析和基因集富集分析考察高表达JOSD2的患者中信号通路的变化情况;利用基因集变异分析和皮尔逊相关性分析考察JOSD2表达水平与DNA损伤反应(DDR)通路的相关性;利用蛋白质印迹法考察JOSD2和DNA损伤修复通路相关蛋白的表达水平;利用免疫荧光法考察JOSD2在细胞内亚定位的变化;利用磺酰罗丹明染色法考察敲低JOSD2对DNA损伤类药物的敏感性。结果:与癌旁组织比较,NSCLC组织中JOSD2的表达量显著上调(P<0.05),且与NSCLC患者的不良预后显著相关(P<0.05);与低表达JOSD2的组织比较,高表达JOSD2的NSCLC组织中DDR相关途径显著激活(均P<0.05),且JOSD2的表达水平与DDR相关途径激活程度呈显著正相关(均P<0.01);与对照组比较,过表达JOSD2显著促进NSCLC细胞系的DDR过程,此外,DNA损伤剂处理可显著提高JOSD2的细胞核定位,而敲低JOSD2显著增强NSCLC细胞对DNA损伤剂的敏感性(均P<0.05)。结论:JOSD2通过促进DNA损伤修复途径调控NSCLC的恶性进展,而敲低JOSD2可显著增强NSCLC细胞对DNA损伤剂的敏感性。

Objective:To investigate the effects and mechanisms of deubiquitinating enzyme Josephin domain containing 2(JOSD2)on susceptibility of non-small cell lung carcinoma(NSCLC)cells to anti-cancer drugs.Methods:The transcriptome expression and clinical data of NSCLC were downloaded from the Gene Expression Omnibus.Principal component analysis and limma analysis were used to investigate the deubiquitinating enzymes up-regulated in NSCLC tissues.Kaplan-Meier analysis was used to investigate the relationship between the expression of deubiquitinating enzymes and overall survival of NSCLC patients.Gene ontology enrichment and gene set enrichment analysis(GSEA)were used to analyze the activation of signaling pathways in NSCLC patients with high expression of JOSD2.Gene set variation analysis and Pearson correlation were used to investigate the correlation between JOSD2 expression levels and DNA damage response(DDR)pathway.Western blotting was performed to examine the expression levels of JOSD2 and proteins associated with the DDR pathway.Immunofluorescence was used to detect the localization of JOSD2.Sulforhodamine B staining was used to examine the sensitivity of JOSD2-knock-down NSCLC cells to DNA damaging drugs.Results:Compared with adjacent tissues,the expression level of JOSD2 was significantly upregulated in NSCLC tissues(P<0.05),and was significantly correlated with the prognosis in NSCLC patients(P<0.05).Compared with the tissues with low expression of JOSD2,the DDR-related pathways were significantly upregulated in NSCLC tissues with high expression of JOSD2(all P<0.05).In addition,the expression of JOSD2 was positively correlated with the activation of DDR-related pathways(all P<0.01).Compared with the control group,overexpression of JOSD2 significantly promoted the DDR in NSCLC cells.In addition,DNA damaging agents significantly increase the nuclear localization of JOSD2,whereas depletion of JOSD2 significantly enhanced the sensitivity of NSCLC cells to DNA damaging agents(all P<0.05).Conclusion:Deubiquitinating enzyme JOSD2 may regulate the malignant progression of NSCLC by promoting DNA damage repair pathway,and depletion of JOSD2 significantly enhances the sensitivity of NSCLC cells to DNA damaging agents.