HPV通过巨噬细胞-NO通路影响口咽癌放疗敏感性的机制研究

HPV infection is one of the critical etiology of oropharyngeal cancer (OPC). The different infection status of HPV include episomal DNA, integrated DNA, transcriptionally active mRNA and transcriptionally inhibitory mRNA. However, there is an obvious difference in the radiosensitivity among patients with HPV+ OPC. The report about the radiosensitivity in different infection status of HPV is not available. In our previous research, we found that OPC patients with episomal HPV were much more sensitive to radiotherapy than those with integrated HPV and had higher nitric oxide (NO) in their tissues. It has been documented that NO could enhance the radiosensitivity of tumor, indicating that the difference in radiosensitivity is related to NO regulated by different infection status of HPV. Our study also showed that in comparison with HPV- OPC, the expression levels of TLR9 and IFNγ were elevated significantly in HPV+ OPC tissue and the amount of macrophage(M1) which could produces abundant NO also increased. Therefore, through bioinformatic analysis, clinical data, cultivating and X-ray radiating tissue in vitro, cultivating mixed cells and CRISPR/Cas9 knockdown experiment, we aim to clarify: 1.The different infection status of HPV can change the radiosensitivity by NO in microenvironment. 2. HPV changes the concentration of NO through TLR9/IFNγ/macrophage(M1) pathway. Our research will reveal a new mechanism of the differences in the radiosensitivityof HPV+ OPC, and provide an experimental evidence and a theoretical basis for precise radiotherapy in patients with HPV+ OPC.

HPV感染是口咽癌的重要病因之一，其感染状态包括游离型/整合型和转录活跃型/抑制型。HPV+口咽癌患者放疗敏感性存在显著差异，但不同感染状态HPV对口咽癌放疗敏感性的影响尚无报道。我们前期发现，HPV游离型口咽癌放疗敏感性优于整合型，且其一氧化氮（NO）浓度较高。已报道NO可增加肿瘤放疗敏感性，提示HPV+口咽癌的放疗敏感性差异与不同感染状态HPV对NO的调节有关。我们还发现，HPV+口咽癌组织中TLR9、IFNγ及可生成高浓度NO的M1型巨噬细胞均增加。因此，本研究拟通过生物信息、临床数据、离体组织培养与X线放射、混合细胞培养、CRISPR/Cas9敲减实验等证明：1.不同感染状态的HPV通过口咽癌微环境NO影响放疗敏感性；2.HPV改变NO浓度的机制是通过TLR9/IFNγ/巨噬细胞M1通路实现的。本研究将揭示HPV+口咽癌放疗敏感差异新机制，为实现口咽癌精准放疗提供理论与依据。

HPV感染是头颈癌的重要病因之一，HPV+头颈癌比HPV-头颈癌放疗敏感，但HPV影响头颈癌放疗敏感的机制有待研究。我们前期发现，HPV+头颈癌组织一氧化氮（NO）浓度较高，NO可增加肿瘤放疗敏感性，提示HPV+头颈癌的放疗敏感可能与NO的调节有关。我们还发现，HPV+头颈癌组织中TLR9、IFNγ及可生成高浓度NO的M1型巨噬细胞均增加，因此我们研究了HPV对”巨噬细胞-NO通路-放疗敏感性”的作用机制。首先，我们对本地区211头颈肿瘤进行免疫组化和PCR检测后明确了本地区头颈癌 HPV感染状态，随后通过免疫荧光法及混合细胞培养等方式发现HPV+头颈癌细胞和组织对放疗更敏感，并且HPV+头颈癌细胞上清可诱导巨噬细胞向M1型极化。利用生物信息学和IHC、多细胞因子检测等方式发现HPV+头颈癌细胞可通过IL-6/IFNγ途径以及外泌体内高水平的Mir-9途径诱导巨噬细胞M1极化，并且M1型巨噬细胞高表达iNOS，合成释放高浓度NO。由于NO可增加头颈癌的放疗敏感性，我们认为不同感染状态HPV通过“HPV- IFNγ/Mir-9-巨噬细胞M1”影响NO产生，进而影响头颈癌放疗敏感性。本研究揭示了HPV+口咽癌放疗敏感差异新机制，为实现口咽癌精准放疗提供了理论与依据。