Anti-cancer effect of miR-139-3p on laryngeal squamous cell carcinoma by targeting rab5a: In vitro and in vivo studies

Abstract

Background: Laryngeal squamous cell carcinoma is the second most common head and neck squamous cell carcinoma. Nowadays, as traditional treatment methods are gradually limited, the development of new treatment methods needs to be resolved. This study aimed to investigate the role of microRNA(miR)-139-3p in laryngeal squamous cell carcinoma, and further explored the underlying mechanism.

Methods: In this study, we first used quantitative real time polymerase chain reaction (qRT-PCR) to detect the level of miR-139-3p in laryngeal squamous cell carcinoma tissue. Then, TargetScan and dual luciferase reporter assay were used to explore and verify whether rab5a was a direct target of miR-139-3p. Thereafter, the expression of miR-139-3p and rab5a in laryngeal squamous cell carcinoma cell line SNU46 was changed by transfection with miR-139-3p mimic or rab5a-plasmid. Then, SNU46 cell proliferation, cell apoptosis, cell cycle, cell migration and cell invasion were determined using Cell Counting Kit-8 (CCK-8), flow cytometry, scratch assay and Transwell assay, respectively. Finally, mouse tumor formation experiments were used to test whether miR-139-3p still exerted its role in inhibiting laryngeal squamous cell carcinoma in vivo.

Results: Compared with the adjacent normal tissues, miR-139-3p significantly down-regulated in laryngeal squamous cell carcinoma tissue. It was confirmed by dual luciferase reporter experiment that rab5a was a direct target of miR-139-3p. Moreover, the up-regulation of miR-139-3p could effectively inhibit the proliferation, migration and invasion of laryngeal squamous cell carcinoma cells, and induced cell cycle arrest and apoptosis. In the molecular level study, we found that up-regulation of miR-139-3p inhibited the expression of rab5a in SNU46 cells. In addition, the protein and mRNA expression of factors related to cell migration, invasion, proliferation and apoptosis, such as integrin β1, Focal adhesion kinase (FAK), paxillin, B cell lymphoma-2 (bcl-2), nuclear factor-kappaB (NF-κB), vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP9), in SNU46 cells were changed after miR-139-3p up-regulation. Consistent with the results of in vitro studies, in vivo experiments showed that miR-139-3p mimic inhibited laryngeal squamous cell carcinoma tumor growth. All the effects of miR-139-3p on laryngeal squamous cell carcinoma were reversed by rab5a over-expression.

Conclusion: miR-139-3p could inhibit laryngeal squamous cell carcinoma by targeting rab5a both in vitro and in vivo.

Keywords: Apoptosis; Invasion; Laryngeal squamous cell carcinoma; Migration; Proliferation; miR-139-3p; rab5a.