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

Abstract

ObjectiveTo 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. MethodsHCT116 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. ResultsThe 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. ConclusionRAI16 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.

Keywords

• colon cancer
• retinoic acid-induced protein 16
• hct116 cells
• phosphoproteomics