磷酸化蛋白质组学联合蛋白质组学分析敲除维甲酸诱导蛋白16对人结肠癌细胞的影响

陈奕孛; 苗根; 王文; 丁翠玲; 戚中田

doi:10.3971/j.issn.1000-8578.2024.24.0351

磷酸化蛋白质组学联合蛋白质组学分析敲除维甲酸诱导蛋白16对人结肠癌细胞的影响

Proteomics and Phosphoproteomics Analysis of Effect of Retinoic Acid-Induced Protein 16 Knockout on Human Colon Cancer Cells

摘要

摘要:
目的分析人结肠癌HCT116细胞敲除维甲酸诱导蛋白16（RAI16）后细胞内总蛋白及磷酸化蛋白质表达的差异，探究RAI16影响HCT116细胞蛋白质功能的可能机制及相关信号通路。
方法收集并提取HCT116 KO和WT细胞蛋白，SDS-PAGE检验蛋白提取效果。利用胰蛋白酶酶解蛋白后，标记肽段并进行质谱分析。对鉴定到的差异蛋白及差异磷酸化蛋白质利用GO数据库、KEGG数据库和STRING数据库进行生物信息学分析。
结果 SDS-PAGE结果提示蛋白无明显降解，且实验组与对照组部分关键条带有明显差异；按Foldchange≥1.5或Foldchange≤1/1.5且P<0.05为条件进行差异蛋白的筛选，共筛选出147个上调差异蛋白和230个下调差异蛋白；并筛选到106个上调磷酸化位点和217个下调磷酸化位点。将去本底差异磷酸化位点功能GO富集分析，发现差异蛋白主要在核质、细胞核及细胞质组成，RNA、钙黏着蛋白及染色质结合，DNA修复、RNA剪接及DNA为模板转录的正调控等多个方面有显著富集趋势。KEGG富集结果显示，差异蛋白主要在核质转运、剪接体、细胞周期、细胞间紧密连接、病毒致癌作用和癌症中的微小RNA等通路具有显著富集趋势。蛋白互作网络主要以 DDX17、NCL、EEF2、CDK1、SSRP1和SMARCC1为核心蛋白质。统计发现，SKP1、ORC1和BAD等两组学差异变化均上调，且磷酸化差异变化比蛋白差异变化更显著，RBL1、RB1、CDK1、CDC6、MCM4、TFDP1、CHD4和SNW1等两组学差异变化均下调，且磷酸化差异变化比蛋白差异变化更显著。
结论 RAI16可能通过SKP1、ORC1、RB1和CDK1等关键蛋白质在多方面生物功能和多条信号通路中发挥作用，影响细胞周期，进而影响癌症发生发展。

Abstract:
Objective To analyze the differences in the expressions of the total and phosphorylated proteins in human colon cancer HCT116 cells after the knockout (KO) of retinoic acid-induced protein 16 (RAI16) and explore the possible mechanism and related signaling pathways affecting its protein function in HCT116 cells.
Methods HCT116 KO and WT cell proteins were collected and extracted, and the protein extraction efficiency was detected via a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment. After protein digestion, the peptides were labeled with TMT and analyzed via mass spectrometry. We used bioinformatics methods to analyze the identified differential proteins and differentially phosphorylated proteins by using GO, KEGG, and STRING databases.
Results The results of SDS-PAGE showed no evident protein degradation. In addition, some key bands were significantly different between the experimental and control groups. A total of 147 up-regulated and 230 down-regulated differential proteins were screened in accordance with the conditions of Foldchange≥1.5 or Foldchange≤1/1.5 and P<0.05. Meanwhile, 106 up-regulated and 217 down-regulated phosphorylation sites were screened. GO enrichment analysis revealed that the differential proteins were mainly enriched in the composition of nucleoplasm, nucleus and cytoplasm, RNA binding, cadherin and chromatin, DNA repair, RNA splicing, and positive regulation of DNA as template transcription. The results of KEGG enrichment indicated that the differential proteins were mainly enriched in nucleocytoplasmic transport, spliceosomes, cell cycle, cell-cell tight junctions, viral carcinogenesis, microRNAs in cancer, etc. The protein interaction network mainly focused on DDX17, NCL, EEF2, CDK1, SSRP1, and SMARCC1. The statistical findings unveiled the up-regulated changes in the two omics of SKP1, ORC1, and BAD and the down-regulated changes in RBL1, RB1, CDK1, CDC6, MCM4, TFDP1, CHD4, and SNW1. Moreover, the phosphorylation differences were more significant than the protein differences.
Conclusion RAI16 plays the possible crucial role in multiple biological functions and signaling pathways through key proteins, such as SKP1, ORC1, RB1, and CDK1, which affect the cell cycle and thereby the occurrence and development of cancer.

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