人乳头瘤病毒16型E6蛋白通过调控miR-27a-3p/SMG1轴增强人乳头瘤病毒阳性头颈部鳞状细胞癌的放射敏感性。

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

作者信息

Long Dan, Xu Li, Deng Zeyi, Guo Dandan, Zhang Yangchun, Liu Zhaohui, Zhang Chunlin

机构信息

Department of Otorhinolaryngology, Head and Neck Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, Guizhou, China.

Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510000, China.

出版信息

Infect Agent Cancer. 2021 Aug 13;16(1):56. doi: 10.1186/s13027-021-00397-w.

DOI:10.1186/s13027-021-00397-w

PMID:34389030

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8361787/

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.

摘要

背景

头颈部鳞状细胞癌（HNSCC）是全球第六大常见恶性肿瘤类型。研究表明，与人乳头瘤病毒（HPV）阴性的患者相比，HPV阳性的HNSCC患者的放射敏感性显著增加。然而，HPV在HNSCC中的临床意义及其调控机制在很大程度上尚不清楚。我们研究的目的是探讨miR-27a-3p在HPV阳性HNSCC细胞放射敏感性中的调控机制。

方法

构建了E6过表达和E6基因敲低的HNSCC细胞系，并通过定量实时PCR（RT-qPCR）和蛋白质免疫印迹法评估转染效率。用E6过表达质粒或E6 siRNA转染后，通过RT-qPCR检测miR-27a-3p和22q11.2微缺失综合征关键区域8（DGCR8）的表达。通过集落形成和TUNEL染色试验探讨miR-27a-3p对HNSCC细胞放射敏感性的影响。使用生物信息学工具和荧光素酶报告基因试验确定SMG1是miR-27a-3p的直接靶标。此外，研究了E6过表达对miR-27a-3p/SMG1轴调控的影响。

结果

在我们的研究中，我们发现HPV E6的过表达上调了HNSCC细胞中DGCR8和miR-27a-3p的表达。接下来，我们证实DGCR8在HNSCC细胞中正向调控miR-27a-3p的表达。荧光素酶报告基因结果证实miR-27a-3p靶向SMG1 mRNA的3'UTR。转染miR-27a-3p模拟物导致SMG1表达降低，而转染miR-27a-3p抑制剂则增加SMG1表达。与对照HNSCC细胞相比，miR-27a-3p模拟物转染的HNSCC细胞的凋亡活性显著增加。在用4 Gy照射处理后，转染miR-27a-3p抑制剂或SMG1过表达质粒的UM-SCC47细胞比相应的对照细胞形成更多的集落。此外，挽救实验表明，HPV16 E6通过靶向miR-27a-3p/SMG1提高了HNSCC细胞的放射敏感性。