HPV16 E6 enhances the radiosensitivity in HPV-positive human head and neck squamous cell carcinoma by regulating the miR-27a-3p/SMG1 axis

Abstract

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is the 6th most common malignant cancer type worldwide. Radiosensitivity has been shown to be significantly increased in patients with human papillomavirus (HPV)-positive HNSCC compared with HPV-negative patients. However, the clinical significance of HPV and its regulatory mechanisms in HNSCC are largely unknown. The aim of our study was to explore the regulatory mechanism of miR-27a-3p in the radiosensitivity of HPV-positive HNSCC cells. Methods E6-overexpressing and E6-knockdown HNSCC cell lines were generated and the transfection efficiencies were evaluated by quantitative real-time PCR (RT-qPCR) and western blotting. The expression of miR-27a-3p and DiGeorge syndrome critical region 8 (DGCR8) was examined by RT-qPCR after transfection with E6 overexpressing plasmid or E6 siRNA. The effects of miR-27a-3p on the radiosensitivity of HNSCC cells were explored by a colony formation and TUNEL staining assays. Bioinformatic tools and luciferase reporter assays were used to identify that SMG1 is the direct target of miR-27a-3p. Furthermore, the effect of E6 overexpression on the regulation of the miR-27a-3p/SMG1 axis was investigated. Results In our study, we found overexpression of HPV E6 upregulated the expression of DGCR8 and miR-27a-3p in HNSCC cells. We next confirmed that DGCR8 positively regulated the expression of miR-27a-3p in HNSCC cells. The luciferase reporter gene results verified that miR-27a-3p targeted the 3’UTR of SMG1 mRNA. MiR-27a-3p mimics transfection resulted in a decrease in SMG1 expression and miR-27a-3p inhibitor transfection increased SMG1 expression. Apoptotic activity of HNSCC cells was significantly increased in miR-27a-3p mimics HNSCC cells compared with control HNSCC cells. After treatment with 4 Gy irradiation, UM-SCC47 cells transfected with miR-27a-3p inhibitor or SMG1 overexpressing plasmid formed more colonies than the corresponding control cells. Furthermore, the rescue experiments demonstrated that HPV16 E6 improved the radiosensitivity of HNSCC cells by targeting miR-27a-3p/SMG1. Conclusion Our study demonstrated that HPV16 E6 activated the DGCR8/miR-27a-3p/SMG1 axis to enhance the radiosensitivity. Our findings might provide a novel therapeutic target to improve the response of HNSCC to radiotherapy.

Keywords

• Head and neck cancer
• HPV16
• Radiosensitivity
• E6
• miR-27a-3p
• SMG1