卵巢癌（OC）是最常见的妇科恶性肿瘤之一。我们先前的研究证实，circNFIX在OC中起着癌基因的作用，能够促进恶性增殖、转移和血管生成。然而，circNFIX在OC免疫逃脱中的作用和机制仍然不清楚。通过qRT-PCR和蛋白质免疫印迹法测定RNA和蛋白质水平，通过细胞计数试剂盒-8、EdU染色、流式细胞术和转移试验测试恶性表型，通过ELISA分析测量免疫细胞因子水平。通过RNA免疫共沉淀、meRIP和双荧光酶方法验证分子相互作用。通过异种移植瘤和肺转移模型进行体内验证。通过苏木精-伊红染色和免疫组织化学染色观察病理变化。circNFIX、PD-L1和IL-6R水平在OC组织和细胞系中上调，而miR-647下调。功能实验显示，circNFIX的丧失抑制了OC细胞的增殖、转移和免疫逃脱。在分子水平上，circNFIX的m6A修饰在OC细胞中上调，并且其表达与m6A修饰呈正相关，依赖于IGF2BP1 ∼ 3的识别。此外，circNFIX作为一个竞争性内源性RNA，通过上调IL-6R表达，从而激活JAK/STAT3信号通路，提高PD-L1表达。救援实验证明，共沉默miR-647可以逆转circNFIX敲除对OC细胞增殖、转移和免疫逃脱的抗肿瘤效应。本研究对于circNFIX在OC中的分子机制提供了综合的理解，证明m6A活化的circNFIX通过调节miR-647/IL-6R/PD-L1通路促进OC的发展和免疫逃脱。版权所有 © 2023 Elsevier B.V. 保留所有权利。

Ovarian cancer (OC) is one of the most common gynecological malignant cancers. Our previous work confirmed that circNFIX acted as an oncogene in OC, which could promote malignant proliferation, metastasis and angiogenesis. However, the role and mechanism of circNFIX in OC immune escape remain unclear.The RNA and protein levels were determined by qRT-PCR and western blot assays. The malignant phenotypes were tested by cell count kit-8, EdU staining, flow cytometry and transwell assays. The immune cytokines levels were measured by ELISA analysis. Molecular interactions were verified employing RNA immunoprecipitation, meRIP and dual luciferase methods. In vivo validation was performed by xenograft tumor and lung metastasis model. Hematoxylin & eosin and immunohistochemistry staining were used to observe the pathological changes.The levels of circNFIX, PD-L1, and IL-6R were upregulated in OC tissues and cell lines, while miR-647 was downregulated. Functional assays showed that loss of circNFIX suppressed the growth, metastasis and immune escape of OC cells both in vitro and in vivo. On the molecular level, the m6A modification of circNFIX was elevated in OC cells, and its expression was positively correlated to m6A modification and depended on IGF2BP1 ∼ 3 recognition. Moreover, circNFIX acted as a competing endogenous RNA for miR-647 to upregulate IL-6R expression, thereby activating JAK/STAT3 signaling and elevating PD-L1 expression. Rescue assays revealed that co-silencing of miR-647 reversed the antitumor effects of circNFIX knockdown on cell proliferation, metastasis and immune escape of OC cells.This study provided a comprehensive understanding of the molecular mechanism about circNFIX in OC, demonstrating m6A activated-circNFIX accelerated OC development and immune escape via regulating miR-647/IL-6R/PD-L1 pathway.Copyright © 2023 Elsevier B.V. All rights reserved.