Nodal通过circRNA000706/miR-143-3p/B3GNT3途径稳定PD-L1蛋白促进 NSCLC免疫抑制的机制研究

Tumor immunosuppression promotes the deterioration of NSCLC; therefore, it is of great significance to reveal related causes and mechanisms, and to find novel potential intervention targets. Nodal is an embryonic protein，which is abnormally and highly expressed in NSCLC. Our preliminary study initially confirmed that Nodal can promote PD-L1 protein expression of NSCLC cells, thereby inhibit the function of T cells. Sequencing data revealed that Nodal up-regulates circRNA000706, and inhibition of circRNA000706 significantly abolishes above mentioned PD-L1 expression. Moreover, circRNA000706 up-regulates the transcription of B3GNT3, while B3GNT3 is proved to be able to stabilize PD-L1 protein. Bio-information data predicts that circRNA000706 may promote the expression of B3GNT3 by adsorption of miR-143-3p. Thus we put forward the potential novel mechanism of immunosuppression in NSCLC that Nodal may promote PD-L1 expression in NSCLC cells via circRNA000706/miR-143-3p/B3GNT3 pathway, then lead to the inhibition of T cell function. In this proposal, we will fully clarify the induction, immunosuppressive effects, as well as the involved key regulatory mechanisms of Nodal on PD-L1 expression, thus provide scientific basis for targeting Nodal and its pathway to relieve NSCLC immunosuppression.

肿瘤免疫抑制促进NSCLC恶性进展，揭示其诱因及机制、寻找潜在干预新靶点意义重大。胚胎期蛋白Nodal在NSCLC中异常高表达。预实验初步证实Nodal可促进NSCLC细胞PD-L1蛋白表达，从而抑制T细胞功能。测序发现Nodal可上调circRNA000706，干扰该circRNA可显著抑制上述PD-L1表达。此外circRNA000706可促进B3GNT3转录，而B3GNT3可稳定PD-L1蛋白；且生信预测circRNA000706可能通过吸附miR-143-3p上调B3GNT3。据此提出引起NSCLC免疫抑制新机制：Nodal通过circRNA000706/miR-143-3p/B3GNT3途径稳定NSCLC细胞PD-L1，引起T细胞免疫抑制。本课题拟充分明确Nodal诱导PD-L1表达的作用及免疫抑制效应，解析关键调控机制，为靶向Nodal及其通路解除NSCLC免疫抑制提供科学依据

研究表明，肿瘤免疫抑制微环境是导致肿瘤发生发展及不良预后的重要因素。程序性死亡配体1（Programmed death-ligand 1, PD-L1）是一种参与肿瘤免疫抑制的重要分子，为程序性死亡受体（Programmed death 1, PD-1）的配体。PD-L1通过与T细胞表面PD-1结合，抑制T细胞活化及功能，从而抑制肿瘤免疫。大量研究表明，肿瘤细胞常通过诱导自身PD-L1表达增加等途径以逃避T细胞免疫杀伤。因此，探讨引起PD-L1表达增加的诱因及其关键机制，寻找解除肿瘤免疫抑制新靶点，已成为当前该领域亟待解决的关键问题。围绕上述关键科学问题，我们展开了以下研究：①我们前期研究发现，Nodal可能通过诱导PD-L1表达，从而介导NSCLC肿瘤免疫抑制。因此，我们进一步从细胞实验、动物实验及临床标本层面展开研究，并发现Nodal可能通过circRNA000706/miR-143-3p/B3GNT3途径促进PD-L1蛋白N-糖基化修饰，从而稳定PD-L1蛋白表达，介导肿瘤免疫逃逸。②我们前期研究发现，在多种肿瘤耐药细胞（耐顺铂治疗的非小细胞肺癌细胞A549/CDDP、耐阿霉素治疗的乳腺癌细胞MCF-7/ADR及肝癌细胞HepG2/ADR）中PD-L1表达显著增加，然而机制不明。我们发现耐药肿瘤细胞中JNK/c-Jun信号通路被显著激活。机制上，COP1/c-Jun/HDAC3信号轴介导的PD-L1启动子区组蛋白H3乙酰化水平增强是维持肿瘤耐药细胞中PD-L1表达增加的关键机制。③EGF/EGFR通路在肝癌组织中异常激活，且与PD-L1蛋白表达紧密相关。因此，我们还进一步探讨了EGF/EGFR通路对肝癌细胞PD-L1表达的影响及关键机制。研究结果发现EGF/EGFR激活可促进肝癌细胞的PD-L1表达从而介导肿瘤免疫逃逸。机制上，EGFR信号通路激活可通过增强p38 MAPK通路活性下调miR-675-5p表达，减少其与PD-L1 3’UTR相结合，从而增强PD-L1 mRNA稳定性，促进PD-L1表达。以上研究结果可为促进肿瘤免疫、提高肿瘤免疫治疗效果提供潜在分子靶点。

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