肾透明细胞癌中核蛋白1对阿昔替尼耐药的作用及机制

Mechanism of nuclear protein 1 in the resistance to axitinib in clear cell renal cell carcinoma

目的:寻找肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)对阿昔替尼耐药的潜在机制,以期拓展对阿昔替尼耐药的理解,便于设计更有针对性的治疗方案,提高患者的治疗效果及生存预后。方法:通过摸索阿昔替尼对ccRCC细胞系786-O与Caki-1的半抑制浓度(half maximal inhibitory concentration,IC_(50)),使用此浓度下的阿昔替尼体外反复刺激细胞30个周期,构建对阿昔替尼耐药的细胞系,未被阿昔替尼处理过的细胞系为敏感细胞系,检测耐药细胞系及敏感细胞系在细胞增殖、细胞凋亡水平上的表型差异。通过转录组测序,在两耐药细胞系共同上调表达的差异基因内筛选出可能参与耐药过程的基因,通过实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)及蛋白质免疫印迹(Western blot,WB)验证耐药细胞系中靶基因的表达量。在基因表达谱交互分析(Gene Expression Profiling Interactive Analysis,GEPIA)数据库中分析靶基因在ccRCC肿瘤及瘤旁组织的表达差异,在卡普兰-梅尔绘图(Kaplan-Meier Plotter,K-M Plotter)数据库中分析靶基因对ccRCC患者的预后影响。对耐药细胞系的靶基因使用慢病毒载体的核糖核酸干扰进行敲低后,再次检测细胞表型差异。使用WB检测不同处理细胞系的细胞凋亡相关蛋白的水平,寻找可能导致耐药的分子通路。结果:体外成功构建出对阿昔替尼耐药的ccRCC细胞系786-O-R与Caki-1-R,其较敏感细胞系的IC_(50)显著升高(分别高出10.99μmol/L,P<0.01;11.96μmol/L,P<0.01)。细胞计数试剂盒-8(cell counting kit-8,CCK-8)、集落形成、5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)实验结果显示耐药细胞系的增殖能力较敏感细胞系下降,但凋亡染色结果显示耐药细胞系的细胞凋亡水平显著降低(P<0.01)。尽管对阿昔替尼耐药,但耐药细胞系在20μmol/L阿昔替尼环境中无明显的新生肿瘤细胞产生。转录组测序筛选出核蛋白1(nuclear protein 1,NUPR1)基因,其核糖核酸(P<0.0001)及蛋白表达水平在耐药细胞系中显著上升。GEPIA数据库分析结果显示NUPR1在ccRCC肿瘤组织中显著高表达(P<0.05);NUPR1高表达的ccRCC患者生存预后更差(P<0.001)。细胞凋亡染色结果显示,敲低NUPR1后抑制了各耐药细胞系对阿昔替尼的抗凋亡能力(786-O,P<0.01;Caki-1,P<0.05)。WB结果显示,敲低NUPR1,被阿昔替尼处理后耐药细胞系的B细胞淋巴瘤(B-cell lymphoma-2,BCL2)蛋白水平下降,BCL2相关X蛋白(BCL2-associated X protein,BAX)水平增加,前体caspase3蛋白表达水平下降,剪切体c-caspase3水平上升。结论:ccRCC细胞系通过NUPR1-BAX/BCL2-caspase3通路减少细胞凋亡,参与了对阿昔替尼的耐药过程。

Objective:To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma(ccRCC),with a view to expanding the understanding of axitinib resistance,facilitating the design of more specific treatment options,and improving the treatment effectiveness and survival prognosis of patients.Methods:By exploring the half maximum inhibitory concentration(IC_(50))of axitinib on ccRCC cell lines 786-O and Caki-1,cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro.Cell lines that were not treated by axitinib were sensitive cell lines.The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested.Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing.The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot(WB).The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis(GEPIA)public database,and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter(K-M Plotter)database.After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector,the phenotypic differences of the cell lines were tested again.WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance.Results:Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro,and their IC_(50) were significantly higher than those of the sensitive cell lines(10.99μmol/L,P<0.01;11.96μmol/L,P<0.01,respectively).Cell counting kit-8(CCK-8)assay,colony formation,and 5-ethynyl-2’-deoxyuridine(EdU)assay showed that compared with the sensitive lines,the proliferative ability of the resistant lines decreased,but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines(P<0.01).Although resistant to axitinib,the resistant lines had no obvious new replicated cells in the environment of 20μmol/L axitinib.Nuclear protein 1(NUPR1)gene was screened by transcriptome sequencing,and its RNA(P<0.0001)and protein expression levels significantly increased in the resistant lines.Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue(P<0.05);the ccRCC patients with higher expression of NUPR1 had a worse survival prognosis(P<0.001).Apoptosis staining results showed that knockdown of NUPR1 inhibited the anti-apoptotic ability of the resistant lines to axitinib(786-O,P<0.01;Caki-1,P<0.05).WB results showed that knocking down NUPR1 decreased the protein level of B-cell lymphoma-2(BCL2),increased the protein level of BCL2-associated X protein(BAX),decreased the protein level of pro-caspase3,and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib.Conclusion:ccRCC cell lines reduce apoptosis through the NUPR1-BAX/BCL2-caspase3 pathway,which is involved in the process of resistance to axitinib.