基于差异蛋白质组学解析紫色杆菌素抑制结肠癌细胞HT29的作用机制

doi:10.3864/j.issn.0578-1752.2017.09.015

摘要/Abstract

摘要： 【目的】对紫色杆菌素作用后的结肠癌细胞HT29进行差异蛋白质组学分析，探究其影响的代谢通路，揭示其抑制癌细胞生长的作用机制，为新型抗癌药物的开发提供一定的参考。【方法】以不同剂量的紫色杆菌素处理HT29 24、48、72 h，通过MTT试验以及透射电镜分析其对结肠癌细胞的有效抑制剂量及抑制情况。在此基础上，对提取的3个处理组的蛋白进行同位素标记，利用反相液相色谱（RP-LC）联用串联质谱（AB SCIEX Triple TOF 5600）获得样品中的多肽及其相对丰度信息，通过数据库（NCBI. Human. protein database）搜索比对，鉴定差异表达蛋白。利用GO分析、KEGG信号通路分析，解析紫色杆菌素抑癌作用代谢途径。【结果】紫色杆菌素对HT29的抑制作用呈一定的时间、剂量依赖性。当紫色杆菌素对HT29的抑制率达50%时，仅为阳性药物5-Fu剂量的1/6，具有更明显的抑制效果。电镜下观察显示，随着紫色杆菌素剂量增大，细胞内部线粒体出现空泡结构，质膜出泡；当浓度达30 mg·L^-1时，细胞膜消失，染色质边集。通过差异蛋白质组学分析，共鉴定出4 258个蛋白，表达差异在2倍以上的蛋白共为757个。其中高剂量组处理差异蛋白数为492个，低剂量组处理的差异蛋白数为112个，阳性对照组差异蛋白数为336个。KEGG分析显示这些差异蛋白共参与50条信号通路。其中有10条信号通路具有显著性（P<0.05），主要参与核糖体循环途径、三羧酸循环途径和RNA降解途径等。【结论】紫色杆菌素主要通过影响HT29细胞生命活动中的蛋白转录和翻译水平进而发挥抑制作用。

关键词: 差异蛋白质组学, 紫色杆菌素, HT29, iTRAQ同位素相对标记和绝对定量, 透射电镜

Abstract: 【Objective】The objective of this experiment is to investigate proteomic profile of HT29 treated with high-dosage-violacein and low-dosage-violacein, and to analyze its involved metabolic pathway and mechanism. 【Method】Cytotoxic activity of violacein was analyzed by MTT assay and observed by using transmission electron microscopy. After that, the total protein was extracted from the cells.To obtain relative abundance of peptides information, total proteins labeled with multiple iTRAQ stable isotopes were segregated and analyzed with RP-LC-MS /MS. The differential expression proteins were identified through NCBI. Human. protein database. The metabolic pathway was analyzed through Go analysis and KEGG analysis. 【Result】 Violacein inhibited the growth of HT29 in dose-dependent and time-dependent manner. Compared with 5-Flu, 5 mg·L^-1 violacein resulted in 50% inhibition of HT29. Its dosage was one sixth of the dosage of 5-Fu. With the increasing violacein, vacuoles in the mitochondrion and membrane blebs were found under transmission electron micrograph. Violet pigment at 30 mg·L^-1 induced margination of nuclear chromatin and disappearance of cell membrane. Through quantitative proteomic analysis of these three cells, 4258 proteins were identified with MS, 757 of which were differential expression proteins (fold change of protein expression≥2). Among these differential expression proteins, there were 492 proteins in cells treated with high dosage of violacein, 112 proteins in cells treated with low dosage of violacein, and 336 proteins in cells treated with 5-Fu. Analysis of enriched KEGG pathway showed that most of these differential expression proteins were involved in 50 signaling pathways, 10 of which were enriched significantly (P<0.05), such as ribosome, citrate cycle and RNA degradation. 【Conclusion】The antitumor mechanism of violacein against colon cancer cells are mainly involved in transcription and translation in cell life cycle.

Key words: comparative proteomics, violacein, iTRAQTM, HT29, transmission electron microscopy

刘鹭，芦晶，王莹，逄晓阳，许嫚，张书文，吕加平. 基于差异蛋白质组学解析紫色杆菌素抑制结肠癌细胞HT29的作用机制[J]. 中国农业科学, 2017, 50(9): 1694-1704.

LIU Lu, LU Jing, WANG Ying, PANG XiaoYang, XU Man, ZHANG ShuWen, Lü JiaPing. Antitumor Effect of Violacein Against HT29 by Comparative Proteomics[J]. Scientia Agricultura Sinica, 2017, 50(9): 1694-1704.

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