MicroRNA-15a/16-mediated cytokine/chemokine reprogramming in Kupffer cells prevents the development of hepatocellular carcinoma

摘要 由于治疗无效，肝细胞癌（HCC）在癌症相关死亡率中排名第三。 肿瘤内积累的调节性T细胞（T细胞），抑制抗肿瘤免疫，已被确定 在HCC。尽管对microRNA在HCC中的作用进行了广泛的研究，但它们在调节免疫抑制中的作用仍然存在。 形容不好。AKT（v-akt鼠胸腺瘤病毒癌基因同源物1）和Nras的异常激活 （神经母细胞瘤ras病毒癌基因同源物）（Ras）在枯否细胞（KCs）和肝细胞（HC）中被观察到 74%的HCC患者。将活化形式的AKT和Nras（AKT/Ras）流体动力学注射（HDI）到小鼠中 在6-8周内引发致命的HCC的发展。miR-15 a和miR-16- 1簇（miR-15 a/16）在来自AKT/Ras小鼠和患者的HCC肿瘤的KC中减少。KC特异性 miR-15 a/16的表达完全阻止了AKT/Ras小鼠中侵袭性HCC的生长，而AKT/Ras小鼠中miR-15 a/16的表达100%阻止了侵袭性HCC的生长。 小鼠在注射后6-8周内死于致死肿瘤负荷。KC特异性表达miR-15 a/16也 导致携带肿瘤的AKT/Ras小鼠中HCC肿瘤的显著消退。机械地，AKT/Ras 将KCs的转录组重编程为KCs的M2极化，并使血清中的 AKT/Ras小鼠KC中C-C基序趋化因子22（CCL 22）及其mRNA水平。相比之下，miR-15 a/16 逆转了这一过程，并通过直接靶向激活转录的NF-κB来抑制CCL 22的过度产生 CCL 22的CCL 22通过CCR 4（C-C趋化因子受体4型）招募TcR。事实上，CCL 22的额外治疗 在AKT/Ras小鼠中恢复了由miR-15 a/16完全预防的HCC的免疫抑制和生长。 我们假设，AKT/Ras训练的KCs通过过度产生CCL 22启动肝脏募集Tcl 3， 从而促进HCC发展;而miR-15 a/16的KC特异性表达抑制肝脏募集 通过减弱CCL 22的产生来抑制THBE和HCC的发展。本项目的目标是阐明 AKT/Ras诱导的KC在促进免疫抑制中的作用，免疫抑制促进HCC的生长，而miR-15 a/16 通过抑制KC中的NF-κB信号转导来逆转这一过程。长期目标是将KC开发为治疗药物， 靶向和miR-15 a/16作为针对HCC的新型免疫疗法。三个具体目标旨在测试我们的 假说.具体来说，我们将（1）揭示AKT/Ras教育的KC促进 免疫抑制和HCC发展。(2)建立miR-15 a/16驱动的机制 （3）评估miR-15 a/16对HCC的治疗潜力， 背景强大的初步数据和逻辑合理的实验设计相结合 使这个项目具有很高的可行性。我们希望这项研究将为免疫抑制提供新的见解， 这有助于设计KCs作为新的治疗靶点和miR-15 a/16作为合理的治疗靶点。 针对HCC的免疫疗法。

Abstract Hepatocellular carcinoma (HCC) ranks the third in cancer-related mortality because of ineffective therapy. Intratumoral accumulation of regulatory T-cells (Tregs), which suppresses antitumor immunity, has been identified in HCC. Despite the extensive research of microRNAs in HCC, their roles in regulating immunosuppression are poorly described. Aberrant activation of AKT (v-akt murine thymoma viral oncogene homolog 1) and Nras (neuroblastoma ras viral oncogene homolog) (Ras) was observed in Kupffer cells (KCs) and hepatocytes (HCs) in 74% of HCC patients. Hydrodynamic injection (HDI) of activated forms of AKT and Nras (AKT/Ras) into mice triggered the development of lethal HCC within 6-8 weeks. MiRNA profiling revealed that miR-15a and miR-16- 1 cluster (miR-15a/16) was reduced in KCs from HCC tumors of AKT/Ras mice and patients. KC-specific expression of miR-15a/16 fully prevented growth of aggressive HCC in AKT/Ras mice, while 100% of AKT/Ras mice died from lethal tumor burden within 6-8 weeks post-injection. KC-specific expression of miR-15a/16 also led to a significant regression of HCC tumors in AKT/Ras mice bearing tumors. Mechanistically, AKT/Ras reprogrammed the transcriptome of KCs toward M2 polarization of KCs and drove a significant increase in serum C-C motif chemokine 22 (CCL22) and mRNA levels of Ccl22 in KCs of AKT/Ras mice. In contrast, miR-15a/16 reversed this process and inhibited CCL22 overproduction by directly targeting NF-κB that activates transcription of Ccl22. CCL22 recruits Tregs via CCR4 (C-C chemokine receptor type 4). Indeed, additional treatment of CCL22 recovered immunosuppression and growth of HCC that was fully prevented by miR-15a/16 in AKT/Ras mice. We hypothesize that AKT/Ras-educated KCs initiate hepatic recruitment of Tregs by overproducing CCL22, thereby promoting HCC development; while KC-specific expression of miR-15a/16 suppresses hepatic recruitment of Tregs and HCC development by attenuating CCL22 production. The objective of this project is to elucidate the role of AKT/Ras-educated KCs in promoting immune suppression that promotes growth of HCC, while miR-15a/16 reverses this process by inhibiting NF-κB signaling in KCs. The long-time goal is to develop KCs as a therapeutic target and miR-15a/16 as a novel immunotherapy against HCC. Three specific aims are designed to test our hypothesis. Specifically, we will (1) unravel the mechanism(s) by which AKT/Ras-educated KCs promote immunosuppression and HCC development. (2) establish the mechanism(s) by which miR-15a/16 drives antitumor immunity; and (3) evaluate the therapeutic potential of miR-15a/16 against HCC with divergent backgrounds. The combination of strong preliminary data and a logical and rationally based experimental design make this project highly feasible. We expect that this study will provide a novel insight into the immunosuppression mechanisms in HCC and facilitate the design of KCs as a novel therapeutic target and miR-15a/16 as a rational immunotherapy against HCC.