BV6诱导人卵巢癌SKOV3细胞凋亡定量蛋白组学研究

Quantitative Proteomics Analysis of BV6 Inducing Apoptosis in Human Ovarian Cancer SKOV3 Cells

目的:通过定量蛋白组学技术探讨Smac类似物BV6影响人卵巢癌SKOV3细胞生长的可能机制。方法:四甲基偶氮唑蓝(MTT)法检测各浓度梯度BV6干预SKOV3细胞48 h后的生长抑制率,计算抑制率为50%时的药物浓度为半数抑制浓度(IC50),以0.05、0.1、0.2μmol/L BV6进行后续实验;光镜下观察对照组与0.05μmol/L BV6处理组48 h后的细胞形态变化,应用同位素标记相对和绝对定量(iTRAQ)技术结合液相串联质谱策略筛选差异蛋白并行生物信息分析;蛋白质印迹(Western blotting)检测对照组、不同浓度BV6处理组半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达。结果:BV6可以抑制SKOV3细胞的生长增殖,且呈剂量依赖性,多组间方差分析及任意组间两两比较,差异均有统计学意义(均P<0.05)。按公式计算BV6作用于SKOV3细胞48 h的IC50为0.40μmol/L。基于发现错误率(FDR)<1%,对照组与0.05μmol/L BV6处理48 h组筛选到349个差异蛋白,其中251个蛋白表达上调、98个蛋白表达下调,经生物信息学分析显示差异蛋白与RNA转录、蛋白质翻译、细胞分裂增殖、凋亡信号调节及肿瘤血管生成密切相关。Western blotting结果显示高、中、低浓度BV6干预SKOV3细胞48 h后与对照组相比,Caspase-3蛋白表达增加,其中高BV6组的Caspase-3蛋白表达最高,组间差异有统计学意义(均P<0.05)。结论:BV6通过抑制RNA转录、蛋白质翻译进程、细胞分裂增殖及肿瘤血管生成,促进Caspase-3凋亡信号活化介导SKOV3细胞死亡。

Objective:To investigate the possible mechanism of Smac mimetic BV6 affecting the growth of human ovarian cancer SKOV3 cells by quantitative proteomics.Methods:MTT assay was used to detect the growth inhibition rate of SKOV3 cells treated with BV6 for 48 hours.Calculate the drug concentration at 50%inhibition rate as half the inhibitory concentration(IC50)and the subsequent experiments were performed at 0.05,0.1,0.2μmol/L BV6.The control group and 0.05μmol/L BV6 treatment group were observed under light microscope,after 48 h the cell morphology was changed.Application of isobarictags for relative and absolute quantitation(iTRAQ)techniques combined with Liquid Chromatography Tandem Mass Spectrometry to Screen Differential Protein Parallel Bioinformatics Analysis;Western blotting was used to detect the expression of caspase-3 in the control group and different concentrations of BV6.Results:BV6 can inhibit the proliferation of SKOV3 cells in a dose-dependent manner,and the variance analysis between groups and the comparison between any two groups were statistically significant(all P<0.05).According to the formula,the IC50 of BV6 acting on SKOV3 cells for 48 h was 0.40μmol/L.Based on FDR<1%,349 differential proteins were screened in the control group and 0.05μmol/L BV6 for 48 h,251 were upregulated and 98 down-regulated.Bioinformatics analysis showed differential protein and RNA transcription and protein.Translation,cell division and proliferation,apoptosis signal regulation and tumor angiogenesis were closely related.Western blotting showed that Caspase-3 protein expression was increased in the high,medium and low concentrations of BV6 for 48 h after treatment with SKOV3 cells.The expression of Caspase-3 protein was highest in the high concentration BV6 intervention group.Significance(all P<0.05).Conclusions:BV6 promotes the death of SKOV3 cells by inhibiting RNA transcription,protein translation,cell division and proliferation,and tumor angiogenesis,and promoting the activation of Caspase-3 apoptosis.