OY-TES-1/miR-326协同调控PD-L1促胶质瘤免疫逃逸的机制初探

Glioma is the most common intracranial malignancy. Immunotherapy has demonstrated its development potential and application prospects in recent years. Our previous study found that cancer-testis antigen OY-TES-1 is not expressed in normal tissues except for testis, and is highly expressed in gliomas. The higher its expression, the worse the prognosis of patients and it may be related to immune tolerance. Preliminary studies have shown that OY-TES-1 and PD-L1 have a common target miR-326. It is known that PD-L1 is an important immune checkpoint. Whether OY-TES-1 acts as a competitive endogenous RNA (CeRNA) to adsorb miR-326, and thus the molecular mechanism regulating PD-L1's involvement in tumor immune escape is worth further studying. In order to prove that OY-TES-1 is the CeRNA of PD-L1, this subject should verify the relationship: (1) Co-expression of OY-TES-1 and PD-L1; (2) OY-TES-1 has miRNA-dependent regulation of PD-L1; (3) OY-TES-1 can affect PD-L1 in the transcriptional and protein levels. Subsequently, functional verification was also performed. An orthotopic xenograft mouse model with dual-fluorescent traces was constructed, then the effects of OY-TES-1/miR-326 coordinated regulation of PD-L1 from tumor cells, tumor microenvironment, and immune cells was observed. This study will help to provide new ideas for tumor immunotherapy and targeted drug selection.

胶质瘤是最常见的颅内恶性肿瘤，近年来免疫疗法已彰显发展潜力和应用前景。我们前期研究发现，癌-睾丸抗原OY-TES-1（OY）在除睾丸外的正常组织几乎不表达，在胶质瘤高表达，其表达越高患者预后越差，与其逃避免疫监控有关。初步研究显示，OY与PD-L1具有共同的靶标miR-326。已知PD-L1是重要的免疫检查点，OY可能作为竞争性内源RNA（CeRNA）吸附miR-326，调控PD-L1参与免疫逃逸，其分子机制值得深究。为证明OY是PD-L1的CeRNA，本课题拟先进行关系验证：⑴OY与PD-L1共表达；⑵OY对PD-L1的调控具有miRNA依赖性；⑶OY能影响PD-L1的转录水平和蛋白水平。其次，进行功能验证：构建双荧光示踪的小鼠原位移植瘤模型，从肿瘤细胞、微环境、免疫细胞等多方面观察OY/miR-326协同调控PD-L1产生的影响，为肿瘤免疫治疗和靶向药物的选择提供新思路。

OY-TES-1作为癌-睾丸抗原家族成员之一，也称ACRBP，因其高表达于胶质瘤等肿瘤而在正常组织（除睾丸外）几乎不表达，被视为较为理想的治疗靶点，但其在肿瘤中的生物学功能和作用机制仍有待研究。本项目从OY-TES-1/miR-326/PD-L1调控网络着手，进行如下研究：.①调控网络的筛选与验证：通过对胶质瘤样本中与OY-TES-1表达相关基因的筛选和下调OY-TES-1后RNA-seq结果，发现OY-TES-1的下游作用分子可能为PD-L1，两者都含有与miR-326的特异性结合位点；经荧光原位杂交、双荧光素酶、RNA免疫共沉淀等实验证实在胶质瘤细胞中OY-TES-1mRNA作为竞争性内源RNA以竞争miR-326方式调控PD-L1表达。.②调控功能的研究：RNA测序提示胶质瘤中OY-TES-1功能与免疫细胞，特别是CD8+T细胞的免疫功能调节有关；体外实验证实OY-TES-1可抑制CD8+T细胞的存活、增殖、细胞因子分泌并促进其凋亡；鼠颅内移植瘤实验进一步揭示OY-TES-1的促瘤能力。.③表达及临床意义分析：应用临床标本结合公共数据库，发现OY-TES-1和PD-L1均高表达于胶质瘤，两者的表达呈正相关、与胶质瘤患者的预后呈负相关，对胶质瘤患者的诊疗及预后具有潜在应用前景；miR-326在胶质瘤中低表达，与OY-TES-1表达呈负相关。.此外，我们的研究还揭示了OY-TES-1也高表达于肝细胞癌和乳腺癌，联用表观遗传药物DAC、VPA和TSA，可增强OY-TES-1特异性T细胞的杀瘤效应。鉴于OY-TES-1在肿瘤中的功能及表达特性，有望成为理想的胶质瘤治疗靶点，具有潜在的临床应用前景。.以项目作为依托，团队2名青年骨干于2021年、2022年首次获得国家自然科学基金资助；3名成员读博深造；3名博士、1名硕士和新进组的1名硕士均顺利毕业。