A comprehensive study of smoking-specific microRNA alterations in head and neck squamous cell carcinoma

Abstract

Objective: While tobacco smoking is a well-known risk factor for head and neck squamous cell carcinoma (HNSCC), the molecular mechanisms underlying tobacco-induced HNSCC remain unclear. This study sought to comprehensively identify microRNA (miRNA) alterations and evaluate their clinical relevance in smoking-induced HNSCC pathogenesis and progression.

Materials and methods: Using small RNA-sequencing data and clinical data from 145 HNSCC patients, we performed a series of differential expression and correlation analyses to identify a panel of tobacco-dysregulated miRNAs associated with key clinical characteristics in HNSCC. We then examined the expression patterns of these miRNAs in normal epithelial cell lines following exposure to cigarette smoke extract.

Results: Our analyses revealed distinct panels of miRNAs to be dysregulated with smoking status and associated with additional clinical features, including tumor stage, metastasis, anatomic site, and patient survival. The differential expression of key miRNAs, including miR-101, miR-181b, miR-486, and miR-1301, was verified in cigarette-treated epithelial cell lines, suggesting their potential roles in the early development of smoking-related HNSCCs.

Conclusion: Specific alterations in miRNA expression may be traced to tobacco use and are associated with important HNSCC clinical characteristics. Future studies of these miRNAs may be valuable for furthering the understanding and targeted treatment of smoking-associated HNSCC.

Keywords: Head and neck neoplasms; MicroRNAs; RNA, Untranslated; Smoking.

Fig. 1

(A) Schematic illustrating the analysis approach used to identify smoking-dysregulated miRNA candidates (p<0.05, FDR < 0.05). (B) Smoking status and miRNA expression patterns. Comparison of miRNA expression between the adjacent normal tissue of HPV(-) lifelong nonsmokers (average of 9 cases, inside circle) and primary tumor of HPV(-) HNSCC current smokers (average of 136 cases, outside circle).

Fig. 2

Boxplots indicating significant variation of (A) smoking-upregulated miRNA expression and (B) smoking-downregulated miRNA expression with anatomic tumor sites (Kruskal-Wallis, p < 0.05).

Fig. 3

Boxplots associating expression of smoking-dysregulated miRNAs to (A) clinical stages, (B) clinical T stage, (C) pathologic T stage and (D) pathologic N stage (Kruskal-Wallis, p < 0.05). In clinical stage analyses, patients with Stage I and II were classified as “Early Stage” and patients with Stage III and IV were classified as “Late Stage.”

Fig. 4

Boxplots correlating expression of smoking-dysregulated miRNAs to (A) perineural invasion (B) lymphocascular invasion and (C) pathological nodal extracapsular spread (Kruskal-Wallis, p < 0.05).

Fig. 5

Kaplan-Meier curves depicting survival outcomes based on relative high and low expression of candidate miRNAs dysregulated by smoking (p<0.05). Hazard ratio with 95% confidence interval are presented for each survival correlation.

Fig 6

qRT-PCR verifies that 0.1% cigarette treatment dysregulates miR-101-3p, miR-101-5p, miR-181b-3p, miR-486-3p, and miR-1301-5p in HaCaT and OKF4. All bar graphs are presented with mean and error bars representing standard deviations. *p< 0.05, **p<0.01, ***p<0.001, Student's t-test.