基于单细胞转录组学的 HPV 阳性头颈部鳞状细胞癌肿瘤异质性探究

背景及目的：头颈鳞状细胞癌（Head and neck squamous cell carcinoma，HNSCC）具有肿瘤异质性，且人乳头瘤病毒（Human papillomavirus，HPV）感染是HNSCC发生的重要原因，与肿瘤的生长、转移等显著相关，但其与HNSCC肿瘤异质性的关系仍不明确。因此，本研究拟利用单细胞转录组测序数据分析法寻找与HPV相关HNSCC异质性及生存相关的关键基因；结合HPV癌基因稳定表达的头颈癌细胞RNA测序数据，明确HPV癌基因对肿瘤异质性的调控机制。

方法：收集5例HPV阳性HNSCC的单细胞转录组测序数据进行肿瘤细胞分群，通过GO功能富集明确各亚群肿瘤细胞的生物学功能；通过拟时序分析探讨各亚群的发育过程的演进；进而分析影响各亚群生物学功能的关键基因。通过CRISPR/Cas9技术构建HPV癌基因 E5、E6、E6*、E7稳定表达的HNSCC细胞系，进行RNA测序并分析癌基因对肿瘤生物功能的影响；通过生物信息学明确HPV癌基因可通过关键基因调控肿瘤亚群。

结果：HPV阳性HNSCC肿瘤存在异质性同时存在转移肿瘤细胞其中干细胞群肿瘤细胞，增殖群肿瘤细胞；干细胞群肿瘤细胞，上皮-间充质转化肿瘤细胞和分化群肿瘤细胞，这两支四类肿瘤细胞异质性明显。HPV E1、E7主要影响干细胞群肿瘤细胞，增殖群肿瘤细胞异质性；E5主要影响干细胞群肿瘤细胞、EMT肿瘤细胞和分化群肿瘤细胞。HPV通过调控肿瘤异质性的相关基因SOX2、HLA-DRA、KRT14、KRT19影响预后。

结论：本研究描述了HPV阳性HNSCC的肿瘤异质性特点，发现HPV主要影响HNSCC肿瘤细胞中干细胞群、增殖细胞群、分化细胞群。HPV E5通过调节HLA-DRA基因的表达改善HNSCC患者的预后。HNSCC细胞拷贝数变异因HPV基因在HNSCC细胞亚群分布的不同而受到影响。

Background and Objectives: Head and neck squamous cell carcinoma (HNSCC) exhibits tumor heterogeneity, with Human papillomavirus (HPV) infection being a significant factor in HNSCC development, closely linked to tumor growth and metastasis. However, the relationship between HPV and tumor heterogeneity in HNSCC remains unclear. This study aims to identify key genes associated with HPV-related HNSCC heterogeneity and survival using ScRNA-seq. By integrating RNA data from head and neck cancer cells with stable expression of HPV oncogenes, the study aims to elucidate the regulatory mechanisms of HPV oncogenes on tumor heterogeneity.

Methods: ScRNA data from 5 cases of HPV-positive HNSCC were collected for tumor cell subtyping. GO functional enrichment was utilized to define the biological functions of tumor cells in each subgroup. Pseudo-time analysis was conducted to explore the developmental progression of each subgroup, followed by the analysis of key genes influencing the biological functions of each subgroup. HNSCC cell lines with stable expression of HPV oncogenes E5, E6, E6*, and E7 were constructed using CRISPR/Cas9 technology for RNA sequencing to analyze the impact of oncogenes on tumor biological functions. Through bioinformatics analysis, the study aimed to clarify how HPV oncogenes regulate tumor subgroups through key genes.

Results: HPV-positive HNSCC tumors exhibit heterogeneity, with subgroups including migrating tumor cells, stem cell group tumor cells, and proliferating tumor cells. Notably, the tumor cells in the stem cell group, Epithelial-Mesenchymal Transition (EMT) tumor cells, and differentiated tumor cells demonstrate significant heterogeneity. HPV E1 and E7 primarily affect the heterogeneity of stem cell group tumor cells and proliferating tumor cells, while E5 mainly influences stem cell group tumor cells, EMT tumor cells, and differentiated tumor cells. HPV regulates tumor heterogeneity through key genes SOX2, HLA-DRA, KRT14, and KRT19, impacting prognosis.

Conclusion: This topic demonstrates the tumor heterogeneity characteristics of HPV+ HNSCC, highlighting the major impact of HPV on stem cell group, proliferating cell group, and differentiated cell group tumor cells. HPV E5 improves the prognosis of HNSCC patients by modulating the expression of the HLA-DRA gene. Copy number variations in HNSCC cells are influenced by the distribution of HPV genes in different subgroups of HNSCC cells.

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