Proteomics and phosphoproteomics analysis of the effect of retinoic acid-induced protein 16 knockout on human colon cancer cells

Objective: The proteomics and phosphorproteomics techniques were used to analyze the differences in the expression of total protein and phosphorylated protein in human colon cancer HCT116 cells after knockout of retinoic acid-induced protein 16 (RAI16), and to explore the possible mechanism and related signaling pathways of RAI16 affecting protein function in HCT116 cells. Methods: HCT116 KO and WT cell proteins were collected and extracted, and the protein extraction efficiency was detected by SDS-PAGE experiment. After digesting the protein, peptides were labeled with TMT and analyzed by mass spectrometry. The identified differential proteins and differentially phosphorylated proteins were analyzed by using GO database, KEGG database and STRING database. Results: The results of SDS-PAGE showed that there was no obvious degradation of protein, and some key bands were significantly different between the experimental group and the control group. A total of 147 up-regulated differential proteins and 230 down-regulated differential proteins were screened according to the conditions of Foldchange≥1.5 or Foldchange≤1/1.5 and p-value <0.05, meanwhile 106 up-regulated phosphorylation sites and 217 down-regulated phosphorylation sites were screened. The GO enrichment analysis showed that the differential proteins were mainly enriched in the composition of nucleoplasm, nuclear and cytoplasmic, the binding of RNA, cadherin and chromatin, DNA repair, RNA splicing and positive regulation of DNA as template transcription. The results of KEGG enrichment showed 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 results showed that the changes of the two omics of SKP1, ORC1 and BAD were up-regulated, and the changes of RBL1, RB1, CDK1, CDC6, MCM4, TFDP1, CHD4 and SNW1 were down-regulated, and the phosphorylation differences were more significant than the protein differences. Conclusion: The proteomics and phosphorproteomics techniques showed that RAI16 may play a crucial role in multiple biological functions and signaling pathways through key proteins such as SKP1, ORC1, RB1 and CDK1, affecting the cell cycle, thereby affecting the occurrence and development of cancer.