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. 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. 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. 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.

中文翻译：

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HPV16 E6通过调节miR-27a-3p/SMG1轴增强HPV阳性人头颈部鳞状细胞癌的放射敏感性

头颈部鳞状细胞癌 (HNSCC) 是全球第六大最常见的恶性肿瘤类型。与 HPV 阴性患者相比，人乳头瘤病毒 (HPV) 阳性 HNSCC 患者的放射敏感性已被证明显着增加。然而，HPV 的临床意义及其在 HNSCC 中的调节机制在很大程度上是未知的。我们研究的目的是探索 miR-27a-3p 在 HPV 阳性 HNSCC 细胞放射敏感性中的调节机制。生成 E6 过表达和 E6 敲低的 HNSCC 细胞系，并通过定量实时 PCR (RT-qPCR) 和蛋白质印迹评估转染效率。在用 E6 过表达质粒或 E6 siRNA 转染后，通过 RT-qPCR 检查 miR-27a-3p 和 DiGeorge 综合征临界区 8（DGCR8）的表达。通过集落形成和 TUNEL 染色分析探讨了 miR-27a-3p 对 HNSCC 细胞放射敏感性的影响。生物信息学工具和荧光素酶报告基因检测被用来鉴定 SMG1 是 miR-27a-3p 的直接靶标。此外，研究了 E6 过表达对 miR-27a-3p/SMG1 轴调节的影响。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。我们接下来证实 DGCR8 正调节 HNSCC 细胞中 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。MiR-27a-3p 模拟转染导致 SMG1 表达降低，而 miR-27a-3p 抑制剂转染增加 SMG1 表达。与对照 HNSCC 细胞相比，miR-27a-3p 模拟 HNSCC 细胞中 HNSCC 细胞的凋亡活性显着增加。用 4 Gy 辐射处理后，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，救援实验表明，HPV16 E6 通过靶向 miR-27a-3p/SMG1 提高了 HNSCC 细胞的放射敏感性。我们的研究表明，HPV16 E6 激活 DGCR8/miR-27a-3p/SMG1 轴以增强放射敏感性。我们的发现可能提供一种新的治疗靶点，以改善 HNSCC 对放疗的反应。用 miR-27a-3p 抑制剂或 SMG1 过表达质粒转染的 UM-SCC47 细胞比相应的对照细胞形成更多的集落。此外，救援实验表明，HPV16 E6 通过靶向 miR-27a-3p/SMG1 提高了 HNSCC 细胞的放射敏感性。我们的研究表明，HPV16 E6 激活 DGCR8/miR-27a-3p/SMG1 轴以增强放射敏感性。我们的发现可能提供一种新的治疗靶点，以改善 HNSCC 对放疗的反应。用 miR-27a-3p 抑制剂或 SMG1 过表达质粒转染的 UM-SCC47 细胞比相应的对照细胞形成更多的集落。此外，救援实验表明，HPV16 E6 通过靶向 miR-27a-3p/SMG1 提高了 HNSCC 细胞的放射敏感性。我们的研究表明，HPV16 E6 激活 DGCR8/miR-27a-3p/SMG1 轴以增强放射敏感性。我们的发现可能提供一种新的治疗靶点，以改善 HNSCC 对放疗的反应。