TAB1——基于蛋白质组学的卵巢癌紫杉醇耐药的潜在生物标志物探讨

TAB1——Potential Biomarker of Paclitaxel Resistance in Ovarian Cancer Based on Label-Free Quantitative Proteomics

目的:探究卵巢癌对紫杉醇(PTX)耐药的潜在靶点及其相关机制。方法:体外培养卵巢癌A2780细胞和A2780PTX耐药细胞(A2780/T),采用不同浓度(2,4,8,16,32,64,128,256μmol·L^(-1))PTX分别干预24 h或48 h,噻唑蓝(MTT)比色法检测PTX对A2780细胞和A2780/T细胞存活率的影响,利用液相色谱质谱/质谱(LC-MS/MS)非标记(Label-Free)定量蛋白质组学方法鉴定和筛选两组细胞中表达差异的蛋白,通过基因本体(GO)注释和京都基因与基因组百科全书(KEGG)通路分析,筛选卵巢癌细胞对PTX耐药的潜在生物标志物。以正常培养的A2780细胞作为空白组,采用0,1,4μmol·L^(-1)的PTX处理A2780/T细胞,采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测验证潜在靶点转化生长因子-β活化激酶1结合蛋白1(TAB1)及其下游相关分子转化生长因子-β活化激酶1(TAK1),p38有丝分裂活化蛋白酶(MAPK)mRNA和蛋白的表达水平。结果:与空白组比较,PTX干预组的A2780和A2780/T细胞的活力均降低。PTX对A2780细胞的抑制率明显高于A2780/T细胞。A2780细胞中,PTX处理48 h的半抑制浓度(IC50)为0.002μmol·L^(-1),A2780/T细胞中,PTX的IC50>设置的最高浓度128μmol·L^(-1),与A2780细胞相比,A2780/T细胞对PTX具有耐药性。通过非标记定量蛋白质组学分析筛选出A2780/T和A2780细胞之间有441种差异表达蛋白和421种特殊差异表达蛋白。GO功能富集分析显示,在差异表达的蛋白质中,结合蛋白占大多数(80%)。根据KEGG通路分析结果和表达位点分析,TAB1被认为可能是卵巢癌对PTX耐药的潜在生物标志物。与A2780细胞比较,A2780/T细胞的TAB1 mRNA和蛋白表达显著降低(P<0.01),与TAB1相互作用的TAK1 mRNA和p38 MAPK mRNA亦明显降低(P<0.05,P<0.01),但蛋白表达无显著变化。结论:TAB1可能是卵巢癌对PTX耐药的潜在生物标志物,其机制可能与TAB1/TAK1/p38 MAPK通路有关。

Objective: To explore the potential targets and related mechanism involved in the paclitaxel resistance to ovarian cancer. Method: Ovarian cancer A2780 cells and A2780 paclitaxel-resistant cells(A2780/T)were treated by 2,4,8,16,32,64,128,256 μmol·L^(-1) paclitaxel(PTX)for 24 h or 48 h respectively in vitro. The proliferation rate of A2780 cells and A2780/T cells treated with paclitaxel was determined by methyl thiazolyl tetrazolium(MTT)colorimetric method assay. A2780 and A2780/T cells were analyzed by LC-MS/MS Label-Free quantitative proteomics to identify and screen differentially expressed proteins in the two groups of cells. Gene ontology(GO)annotation and Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were used to determine the potential biomarkers of paclitaxel resistance in ovarian cancer. Conventionally cultured A2780 cells were used as a control group,and A2780/T cells were treated with 0,1,4 μmol·L^(-1) PTX. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot methods were used to detect and verify the m RNA and protein expression levels of potential target transforming growth factor-β-activated kinase 1 binding protein 1(TAB1)and its downstream related molecules transforming growth factor-β-activated kinase(TAK1)and p38. Result:After PTX treatment for 24 h and 48 h,the cell viability of A2780 and A2780/T cells decreased. The inhibitory rate of PTX on A2780 cells was significantly higher than that of A2780/T cells. In A2780 cells,the IC50 of PTX treatment for 48 h was0.002 μmol·L^(-1),while in A2780/T cells,the IC50 of PTX was greater than the maximum concentration of 128μmol·L^(-1),indicating that A2780/T cells were resistant to PTX compared with A2780 cells. 441 differentially expressed proteins and 421 special differentially expressed proteins between A2780/T and A2780 cells were screened by label-free quantitative proteomic analysis. GO function enrichment analysis showed that the binding proteins accounted for the majority(80%)among the differentially expressed proteins. According to the results of KEGG pathway analysis and expression site analysis,TAB1 might be a potential biomarker in paclitaxelresistant ovarian cancer. Compared with A2780 cells,m RNA and protein expression levels of TAB1 in A2780/T cells were significantly reduced(P<0.01). m RNA expression of TAK1 and p38 that interacted with TAB1 were also significantly reduced(P<0.05,P<0.01),while there was no significant change in protein expression.Conclusion: TAB1 may be a potential biomarker of paclitaxel resistance to ovarian cancer,and its mechanism may be related to the TAB1/TAK1/p38 MAPK pathway.