Screening Differentially Expressed Proteins in Areca Nut-Related Oral Squamous Cell Carcinoma Using Tandem Mass Tag Proteomics

Abstract

Objective: Areca nut chewing has been associated with a poor prognosis in oral squamous cell carcinoma (OSCC). This study seeks to identify differentially expressed proteins among areca nut-related OSCC,non-areca nut-related OSCC and adjacent normal epithelial tissues, with the aim of providing novel insights for the investigation of areca nut-related OSCC.

Methods: Using Tandem mass tag (TMT)-based proteomic analysis, a comparative proteomic profiling was conducted among areca nut-related OSCC, non-areca nut-related OSCC, and adjacent normal epithelial tissues (n=15). GO and KEGG enrichment analyses were then employed to identify significant proteins pertinent to the pathogenesis of OSCC for further study. Western Blot (WB) and Immunohistochemistry (IHC) techniques were used to preliminary validate the expression patterns of LZTS1, MMP10, MYH6, MB, and TNNC1 among various groups (n=30).

Results: 27 differentially expressed proteins were identified when comparing the areca nut-related OSCC group with both the non-areca nut-related OSCC and normal epithelial tissue groups. Among these, 15 proteins were upregulated, while 12 were downregulated. LZTS1 and MMP10 were included in the upregulated proteins, whereas MYH6, MB, and TNNC1 were downregulated. WB and IHC analyses corroborated the proteomic findings, revealing consistent expression trends for these 5 proteins across the studied groups.

Conclusion: LZTS1, MMP10, MYH6, MB and TNNC1 emerge as promising biomarkers for assessing disease progression, prognosis, and potential targeted therapies in areca nut-related OSCC.

Keywords: Areca nut; Biomarkers; Oral squamous cell carcinoma (OSCC); Proteomics; Tandem mass tag (TMT).

Fig. 1

TMT labeling information. No.: Experiment number for multiple group labeling.

Fig. 2

Protein Identification Results Statistics (n=15).

Fig. 3

Volcanic diagram of arec nut-related OSCC group (A)(n=5) VS normal group (C)(n=5). Red dots represent up-regulated proteins, blue dots represent down-regulated proteins.

Fig. 4

Volcanic map of areca nut-related OSCC group (A)(n=5) VS non-areca nut-related OSCC group (B)(n=5). Red dots represent up-regulated proteins, blue dots represent down-regulated proteins.

Fig. 5

Venn diagram. This Venn diagram illustrates the differentially expressed proteins obtained from pairwise comparisons between the areca nut-related oral squamous cell carcinoma (OSCC) group (A)(n=5), the non-areca nut-related OSCC group (B)(n=5), and the normal oral mucosal tissue group (C)(n=5). Each region in the diagram represents the shared or unique differentially expressed proteins among the 3 groups, providing a visual representation of the distribution and overlap of differentially expressed proteins between the groups.

Fig. 6

GO enrichment bubble map. The vertical axis represents GO function names, while the horizontal axis displays the significance P-values (transformed using -Log10). In this plot, blue signifies Biological Process (BP), yellow denotes Molecular Function (MF), and red represents Cellular Component (CC). The size of the circles corresponds to the number of differentially expressed proteins associated with each GO function.

Fig. 7

KEGG enrichment bubble map. The vertical axis represents pathway names, while the horizontal axis displays the Rich Factor values. The circle colors indicate the enrichment significance P-values (-Log10 transformed). Darker colors correspond to smaller P-values, indicating higher statistical significance. Larger circle sizes represent a greater number of proteins contained within the pathway.

Fig. 8

WB validation results for DEPs. The expression patterns of LZTS1, MMP10, MYH6, MB, and TNNC1 in areca nut-related OSCC(A)(n=10), non-areca nut-related OSCC(B)(n=10), and the normal oral mucosal tissue group(C)(n=10) are consistent with the proteomics results.

Fig. 9

IHC of LZTS1 and MMP10 in each group (100 ×; 200 ×). LZTS1 and MMP10 are highly expressed in areca nut-related OSCC group; (Group A: areca nut-related OSCC (n=10); Group B: non-areca nut-related OSCC (n=10); Group C: normal oral mucosal tissue group) (n=10).

Fig. 10

IHC of MYH6, MB and TNNC1 in each group(100 ×; 200 ×). MYH6, MB and TNNC1 are highly expressed in the normal oral mucosal tissues. (Group A: areca nut-related OSCC (n=10); Group B: non-areca nut-related OSCC (n=10); Group C: normal oral mucosal tissue group) (n=10).