GADD45A与POLE3介导基因组不稳定性重塑宫颈癌免疫微环境的机制

Mechanism of genomic instability mediated by GADD45A and POLE3 to reconstruct immune microenvironment of cervical cancer

摘要

目的：探讨GADD45A与POLE3在宫颈癌中的表达特征及其对细胞生物学行为和患者预后的影响。方法：采用免疫组织化学染色（IHC）检测69例宫颈癌组织及23例正常宫颈组织中GADD45A与POLE3的蛋白表达水平,在HeLa与SiHa细胞系中分别构建oeGADD45A(过表达)及siPOLE3（敲低）模型，通过CCK‐8法、平板克隆形成实验、流式细胞术评价细胞增殖、凋亡及周期的变化。利用转录组测序分析二者调控的差异表达基因集及富集通路，并基于TCGA数据库评估其与患者总生存期的相关性。结果：IHC显示宫颈癌组织中GADD45A表达显著下调，而POLE3表达显著上调，且在HPV16阳性组中升高尤为明显（P<0.05）。功能实验显示，oeGADD45A或siPOLE3均可显著抑制宫颈癌细胞增殖并诱导细胞凋亡（P<0.05）；周期分析表明oeGADD45A可引起S期阻滞；测序分析揭示oeGADD45A与siPOLE3诱导的转录组变化高度相似，差异基因主要富集于核小体组装、DNA损伤应答及干扰素信号通路；TCGA生存分析显示，POLE3高表达与宫颈癌患者的不良预后显著相关（P<0.01），而GADD45A高表达预示患者生存获益。结论：GADD45A在宫颈癌中发挥抑癌作用，而POLE3为促癌因子；二者可能通过共同调控DNA损伤修复与免疫应答通路影响宫颈癌进展，可作为临床预后评估及靶向治疗的潜在标志物。

Abstract

Objective: To investigate the expression of GADD45A and POLE3 in cervical cancer and their effects on cell biological behavior and prognosis of patients. Methods: The expression levels of GADD45A and POLE3 protein in 69 cervical cancer tissues and 23 normal cervical tissues were detected by immunohistochemical staining (IHC). oeGADD45A and siPOLE3 models were constructed in HeLa and SiHa cell lines, respectively. Cell proliferation, apoptosis and cell cycle were evaluated by CCK‐8 assay, colony formation assay and flow cytometry. Transcriptome sequencing was used to analyze the differentially expressed gene sets and enriched pathways regulated by the two, and their correlation with the overall survival of patients was evaluated based on TCGA database. Results: IHC results showed that GADD45A expression was significantly down‐regulated, while POLE3 expression was significantly up‐regulated in cervical cancer tissues, especially in the HPV16 positive group (P<0.05). Functional experiments showed that oeGADD45A or siPOLE3 significantly inhibited the proliferation and induced apoptosis of cervical cancer cells (P<0.05). Cell cycle analysis showed that oeGADD45A could induce S phase arrest. Sequencing analysis revealed that the transcriptome changes induced by oeGADD45A and siPOLE3 were highly similar, and the differential genes were mainly enriched in nucleosome assembly, DNA damage response and interferon signaling pathway. TCGA survival analysis showed that high expression of POLE3 was significantly associated with poor prognosis of cervical cancer patients (P<0.01), while high expression of GADD45A predicted survival benefit of patients. Conclusion: GADD45A plays a tumor suppressor role and POLE3 is a tumor promoter in cervical cancer. They may affect the progression of cervical cancer by jointly regulating DNA damage repair and immune response pathways, and can be used as potential markers for clinical prognosis evaluation and targeted therapy.

关键词

宫颈癌 / GADD45A / POLE3 / 人乳头瘤病毒 / 免疫微环境 / 免疫检查点

Key words

Cervical Cancer / GADD45A / POLE3 / Human Papillomavirus / Immune Microenvironment / Immune Checkpoint

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基金资助

国家自然科学基金面上项目(82473294)