衰老的神经母细胞瘤通过外泌体miR-126介导抑制CTL和NK细胞活性导致免疫逃逸的机制研究

Neuroblastoma is the most common extracranial solid tumor of children. Patients with high-risk neuroblastoma have very poor outcome despite intensive therapy. Chemotherapy is still a major therapy, but it only delays the tumor growth. In addition to apoptosis, chemotherapy also induces cellular senescence. Inducing senescence is an alternative therapy because it can also delay tumor regression with low dose therapy, which is extremely important for children cannot tolerate high-dose therapy. Since senescent tumor cells have many DNA mutations due to DNA damage by therapy, these senescent tumor cells have a high chance to re-enter into cell cycle leading to tumor recurrence. Clearing senescent tumor cells can reduce the chance of tumor recurrence. Recent studies showed that senescent cells will secret a lot of cytokines and chemokines to recruit and activate some immune cells, including cytotoxic T lymphocytes（CTL） and NK cells, to clear these senescent cells. Our preliminary data showed that although there were a lot of CTL and NK cells in the senescent tumor tissues, their activity were much lower than those in non-senescent tumor tissues and those in blood. Therefore, their activity was inhibited by some factors in the senescent tumor tissues. Recent studies indicate that tumor exosomes can affect immune cells in the tumor microenvironment and lead to tumor immune escape. We analyzed the miRNAs in the exosomes secreted by therapy-induced senescent neuroblastoma cells and found that the levels of some miRNAs were much higher than those in exosomes of non-senescent neuroblastoma cells. In addition, we observed that these miRNAs inhibited the activity of immune cells. We further studied each individual miRNA, and found that miR-126 had the most influence of immune cells, which let us hypothesized in the first section of this proposal that high levels of miR-126 secreted by therapy-induced senescent neuroblastoma cells reduced the activity of tumor killing immune cells, leading to tumor immune escape. Based on bioinformatics prediction, miR-126 may target mRNAs of glutamate receptors. Since glutamate serves as the precursor of glutamine and glutamine is essential for metabolisms, we predicted in the second section of this proposal that miR-126 targeted glutamate receptors in immune cells and reduced metabolisms, resulting in inhibition of proliferation and activity of immune cells in the senescent tumor microenvironment. Since based on our preliminary data and other studies that senescent tumor cells have higher expression of TRAIL receptor and TRAIL can induce apoptosis in senescent tumor cells, in the third section of this proposal, we will explore some combination therapy to induce apoptosis in therapy-induced senescent neuroblastoma cells to reduce tumor recurrence. Overall, this study will help us further understand immune escape related tumor recurrence and may lead to new therapy to reduce neuroblastoma recurrence and drug resistance.

高危神经母细胞瘤预后不良，化疗仅暂时缓解。化疗除诱导凋亡外，同时诱导肿瘤细胞衰老。衰老相关分泌表型含很多细胞因子和趋化因子，吸引细胞毒T淋巴细胞(CTL)和自然杀伤细胞(NK)聚集，以清除衰老肿瘤细胞，但仍有衰老细胞逃逸免疫。我们前期发现，衰老的肿瘤细胞以外泌体的形式分泌大量miR-126，导致CTL和NK细胞活性下降。本项目利用细胞系和动物模型，探讨化疗诱导的衰老肿瘤细胞分泌的miR-126是否与抑制CTL和NK细胞活性相关；采用生物信息技术预测miR-126靶基因为谷氨酸受体；利用细胞转染和质谱分析技术，研究miR-126是否通过下调免疫细胞谷氨酸受体及相关氨基酸代谢来抑制CTL和NK细胞活性；探索化疗联合免疫激活剂或肿瘤坏死因子相关凋亡诱导配体（TRAIL）能否清除衰老的神经母细胞瘤细胞，降低复发风险。研究结果将进一步阐明神经母细胞瘤复发机制，为探索新的治疗理念提供重要实验基础。

神经母细胞瘤是儿童最常见的颅外恶性实体肿瘤，大多数年龄18月以上的患儿预后很差，早期即可发生远处转移。化疗可同时诱导肿瘤细胞凋亡和衰老，有研究显示，未被清除掉的衰老肿瘤细胞很有可能是肿瘤复发的原因之一，衰老逃逸的肿瘤细胞会再次进入细胞周期，并具备更强的增殖和侵袭能力。本项目主要研究了小剂量化疗药诱导神经母细胞瘤细胞衰老后衰老相关通路的变化及与NKG2D配体相关的免疫逃逸机制。研究利用了神经母细胞瘤细胞系，采用了衰老相关的半乳糖苷酶（SA-β -gal）染色、Western blot、RT-PCR、siRNA、外泌体提取、ELISA等实验方法。根据实验结果我们发现，小剂量化疗药MLN8237诱导SK-N-SH细胞衰老激活了P53/P21通路，P53的升高又引起miR-34a-5p的上调，进而导致其靶基因SIRT1表达降低，而SIRT1的降低又使P53降解减少，进一步促进P53表达的升高，以此形成一个P53/miR-34a-5p/SIRT1反馈环路在MLN8237诱导的细胞衰老中发挥作用。此外，不同的神经母细胞瘤细胞系化疗诱导细胞衰老后可刺激含有MICA/B的外泌体释放，抑制免疫细胞NKG2D受体的表达，衰老的同时金属蛋白酶ADAM10上调，引起IMR-32细胞表面NKG2D受体脱落。将ADAM10抑制剂GI254023X与化疗药物联用，可抑制MICA/B分子的分泌，将其保留在细胞表面，增强免疫细胞的识别能力。进一步研究发现LncRNA MALAT1通过竞争性抑制调节miR-92a，从而上调金属蛋白酶ADAM10的表达，胞内转染siRNA抑制MALAT1可下调MICA/B的分泌，增强NK细胞识别及杀伤功能，联合抑制miR-92a可逆转此现象。本项目关于神经母细胞瘤细胞衰老分子机制的研究可为利用细胞衰老治疗癌症提供新的理论基础，免疫逃逸机制可为神经母细胞瘤的治疗提供参考。