雷公藤甲素诱导人肺癌细胞A549凋亡的iTRAQ定量蛋白质组学研究

发布时间：2018-04-27 16:41

本文选题：雷公藤甲素 + 肺癌　；参考：《浙江中医药大学》2017年硕士论文

【摘要】：目的研究雷公藤甲素对人肺癌A549细胞系蛋白质表达谱的影响,并对差异表达的蛋白质进行GO功能注释、KEGG通路富集和蛋白质相互作用网络构建(PPI),探讨雷公藤甲素诱导肺癌细胞A549凋亡的潜在作用靶点和分子机制。方法1、以肺癌细胞A549为研究对象,CCK-8实验筛选雷公藤甲素(6.25、12.5、25、50、100、200和400 ng/ml)作用于A549细胞后抑制细胞增殖的最佳作用浓度和时间。2、流式细胞术检测雷公藤甲素(12.5、50、200 ng/mL)对肺癌A549细胞周期和细胞凋亡的影响。3、采用体外iTRAQ标记结合纳升液相色谱串联质谱(NanoLC-MS/MS)的定量蛋白质组学技术,并利用PrOteinPi1Ot4.5软件筛选雷公藤甲素作用前后肺癌细胞A549中的差异表达蛋白;并对差异表达蛋白进行GO功能注释、KEGG通路富集和蛋白质相互作用网络(PPI)构建,预测雷公藤甲素抗肿瘤作用的潜在靶点和分子机制。4、通过Western blot方法验证部分差异蛋白(PHB、CDH1、AIFM1、MTA2和EIF4A3)在肺癌细胞A549中的表达情况。结果1.雷公藤甲素能够剂量依赖性地抑制肺癌A549细胞增殖,24h和36h的IC50分别是273ng/mL和210ng/mL。雷公藤甲素(12.5、50、200ng/mL)能以剂量依赖性方式阻滞人肺癌细胞A549细胞周期于G2/M期(P0.01),并诱导其发生凋亡(P0.01)。实验采用200ng/mL的雷公藤甲素和36h作用时间进行后续的蛋白质组学实验。2.运用iTRAQ定量蛋白质组学技术筛选雷公藤甲素处理前后人肺癌细胞A549内的差异蛋白结果显示:雷公藤甲素组与空白对照组相比,雷公藤甲素干预可以引起A549细胞中的312种蛋白质发生显著的差异性表达(P0.05),其中141个蛋白质表达上调,171个蛋白质表达下调。生物信息学分析表明这些蛋白质参与多种生物学途径,包括真核生物核糖体的生物合成,RNA加工,核糖核蛋白复合物生物合成,rRNA代谢,rRNA加工,ncRNA加工,细胞组分生物合成等,涉及226种不同的KEGG通路并且彼此相互联系构成网络。5.Western Blot验证实验结果显示:在人肺癌细胞A549中,雷公藤甲素可以上调PHB、CDH1和AIFM1蛋白质表达(P0.05),下调MTA2和EIF4A3蛋白质表达(P0.05),与蛋白质组学结果一致。结论1.雷公藤甲素(200ng/mL,36h)对A549细胞有显著的细胞毒性作用,可以抑制细胞增殖,显著阻滞细胞周期于G2/M期并诱导细胞凋亡(P0.01)。2.雷公藤甲素可以影响肺癌细胞A549内的多种生物学途径,包括真核生物中的核糖体生物合成,剪接体,mRNA监控途径,PARP1/AIF途径,代谢途径,上皮间质转化(EMT)和其他重要的分子靶标等,这些途径很可能是雷公藤甲素抗肿瘤作用的有效靶点。
[Abstract]:Objective to study the effect of triptolide on protein expression profile of human lung cancer cell line A549. The differentially expressed proteins were enriched in the KEGG pathway and constructed by protein interaction network to explore the potential target and molecular mechanism of triptolide induced apoptosis in lung cancer cell line A549. Methods 1. The best concentration and time of triptolide on A549 cells was selected by CCK-8 assay. Flow cytometry was used to detect the effect of triptolide on A549 cell line. Flow cytometry was used to detect the effect of triptolide (12.5ngmL) on A549 lung cancer cell line (A549 cells) treated with triptolide (6.25ng / ml) and 400ng / ml respectively. Flow cytometry (FCM) was used to detect the effect of triptolide (12.5ngmL) on lung cancer cell line A549. The effects of cell cycle and apoptosis on cell cycle. 3. Quantitative proteomics technique using in vitro iTRAQ labeling combined with NanoLC-MS / MS (NanoLC-MS / MS) was used. PrOteinPi1Ot4.5 software was used to screen differentially expressed protein in lung cancer cell line A549 before and after treatment with triptolide, and to construct the differential expression protein, which was enriched by go functional annotated KEGG pathway and constructed by protein interaction network. The potential target and molecular mechanism of triptolide were predicted, and the expression of partial differential proteins (PHB-CDH1, AIFMTA2 and EIF4A3) in lung cancer cell line A549 was confirmed by Western blot method. Result 1. Triptolide inhibited the proliferation of lung cancer A549 cells in a dose-dependent manner. The IC50 of 24 h and 36 h were 273ng/mL and 210 ng / mL, respectively. Tripterygium wilfordii 12.5ng / mL could block the cell cycle of human lung cancer cell A549 in a dose-dependent manner and induce apoptosis in A549 cells in G _ 2 / M phase. 200ng/mL triptolide and 36 h action time were used to carry out the subsequent proteomics experiment. 2. ITRAQ quantitative proteomics was used to screen the differentially expressed proteins in human lung cancer cell line A549 before and after triptolide treatment. Tripterygium wilfordii could induce 312 proteins in A549 cells to be significantly differentially expressed, of which 141 proteins were up-regulated and 171 proteins were down-regulated. Bioinformatics analysis shows that these proteins are involved in many biological pathways, including the biosynthesis of eukaryotic ribosomes, the biosynthesis of ribosomal protein complexes, the metabolism of rRNA and the processing of ncRNA, and the biosynthesis of cell components. Western Blot verification results show that in human lung cancer cell line A549, there are 226 different KEGG pathways involved and they are connected with each other to form a network. 5. Western Blot verification results show that in human lung cancer cell line A549, Tripterygium wilfordii can up-regulate the expression of AIFM1 and CDH1 protein and down-regulate the expression of MTA2 and EIF4A3 protein, which is consistent with the results of proteomics. Conclusion 1. Tripterygium wilfordii (200ng / mL for 36h) has a significant cytotoxic effect on A549 cells, which can inhibit cell proliferation, significantly block cell cycle at G _ 2 / M phase and induce apoptosis (P _ (0.01) P _ (0.01) 路2). Triptolide can affect many biological pathways in lung cancer cell A549, including ribosomal biosynthesis in eukaryotes, splicing mRNA monitoring pathway, PARP1 / AIF pathway, metabolic pathway, epithelial mesenchymal transformation (EMTT) and other important molecular targets. These pathways may be an effective target for triptolide's anti-tumor effect.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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