Bioinformatics and statistical analysis

The functional characteristics of the identified UPs, including biological processes (BPs), molecular functions (MFs), and cellular components (CCs) were analyzed with DAVID software (version 6.8, https://david.ncifcrf.gov/) (25). Ubiquitination-mediated signaling pathways were analyzed with the KEGG tool KOBAS (http://kobas.cbi.pku.cn) (26). The protein-protein interaction (PPI) networks that UPs are involved in were analyzed with STRING software (https://string-db.org/; version 10.0) (27), with statistical significance of a combined score > 0.4, in the Cytoscape program (version 3.6.0) (28). Also, molecular complex detection (MCODE) in the Cytoscape program was used to identify significant modules in the PPI networks with cutoff values (max depth=100, node score=0.2, degree=2, k-score=2, and MCODE score>5) (29). The hub nodes of PPI network were determined with cytoHubba (version 0.1) in Cytoscape package through 6 different topological algorithms (30), including Maximal Clique Centrality (MCC; cutoff score=100 with this node that had at least 5 molecules linked), Maximum Neighborhood Component (MNC; cutoff score=10 with this node that had at least 5 molecules linked), Degree (cutoff score=10 with this node that had at least 5 molecules linked), Edge Percolated Component (EPC; cutoff score=10 with this node that had at least 5 molecules linked), Closeness (cutoff=150 with this node that had at least 5 molecules linked), and Raliality (cutoff score=5 with this node that had at least 5 molecules linked). The final hub nodes were identified with overlapped analysis of six sets of hub nodes that were derived from 6 topological algorithms. The transcriptomic data (level 2 count data) and corresponding clinical survival data of LSCC patients were obtained from TCGA database with R-languages “RTCGA.mRNA” (version 1.12.0) and “RTCGA.clinical” (version 20151101.14.0), respectively. The survival analysis was performed with R-languages “survival” (version 2.44-1.1) and “survminer” (version 0.4.6).

The overall flow-chart to identify and analyze the ubiquitination profile and the corresponding functional characteristics in LSCC was summarized ( Figure 1 ).

Experimental flow-chart and bioinformatics analysis that were used in this study.