肝癌TACE术后缺氧微环境HIF-1α调控PD-L1过表达介导肿瘤免疫逃逸的作用及机制研究

Hypoxia plays an important role on malignant tumor immune escape, which has close relationship with the overexpression levels of hypoxia inducible factor-1α (HIF-1α) and programmed death-ligand 1 (PD-L1). We had reported that HIF-1α could be overexpressed in hypoxic hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE) treatment. However, the effects and mechanisms of HIF-1α regulated overexpression of PD-L1 on HCC immune escape in hypoxic microenvironment after TACE are still uncertain. Therefore, our study is urgently needed to validate this scientific hypothesis. In vitro study, the effects and mechanisms of HIF-1α regulated overexpression of PD-L1 on immune escape of hepatoma cells will be firstly studied in hypoxic microenvironment. In vivo study, after the treatment of TACE for HCCs of rats, the specific siRNA compound will be transfected into tumor tissues to silence the expressions of HIF-1α and PD-L1. After the combined treatment for HCCs of rats, the local immune functions of tumor microenvironment (infiltration level, apoptosis and cytolytic activity of CD8+T lymphocytes) and the change of biological behavior (proliferation, apoptosis and invasion activity) of hepatoma cells were detected to investigate the effects and mechanisms of HIF-1α regulated overexpression of PD-L1 on HCC immune escape in hypoxic microenvironment after TACE. Finally, the findings of this study will provide reliable scientific basis to improve the long-term outcomes of TACE in the treatment of HCCs.

缺氧是恶性肿瘤发生免疫逃逸的重要原因，与缺氧诱导因子-1α(HIF-1α)和程序性死亡受体-配体1(PD-L1)过表达密切相关。本组已证实：肝动脉化疗栓塞(TACE)术后缺氧肝癌细胞可过表达HIF-1α。但TACE术后缺氧环境HIF-1α能否调控PD-L1过表达介导肿瘤免疫逃逸，机制如何，尚无相关报道，亟待立项研究。本项目拟首先在体外缺氧环境明确肝癌细胞基于HIF-1α和PD-L1过表达的细胞毒性T淋巴细胞免疫逃逸发生机制。后续动物实验拟转染特异性siRNA于TACE术后的大鼠肝癌组织，沉默HIF-1α和PD-L1表达。检测肿瘤局部免疫功能状态(CD8+T细胞浸润程度、凋亡情况及细胞毒性等)和肿瘤细胞生物学行为(增殖、凋亡及侵袭等)发生的变化。阐明肝癌TACE术后缺氧微环境HIF-1α调控PD-L1过表达介导肿瘤免疫逃逸的作用及机制，为突破TACE治疗肝癌远期疗效欠佳的瓶颈奠定理论基础。

肝动脉化疗栓塞术（TACE）是中晚期肝细胞癌（HCC）的首选治疗手段。缺氧肿瘤细胞PD-L1与HIF-1α表达水平密切相关。然而，TACE术后HCC缺氧加剧能否导致PD-L1表达升高，机制如何，有待进一步研究。本研究主要探讨缺氧调控肝癌PD-L1过表达，促进肿瘤进展的作用及分子机制。.离体条件下，我们证实缺氧可通过上调PD-L1、HIF-1α、TCF 4、LEF 1、VEGF、MMP 9、SNAIL及CCND 1表达，促进肝癌细胞进展。荧光素酶活性检测显示缺氧肝癌细胞Wnt /β-catenin信号通路被激活。XAV939抑制Wnt /β-catenin信号通路激活后，PD-L1蛋白、细胞核内β-catenin蛋白表达水平明显下降（P﹤0.05）、HIF-1α蛋白表达水平未发生明显变化（P >0.05）。HIF-1α基因表达沉默（siRNA）后，HIF-1α、PD-L1及细胞核内β-catenin蛋白表达水平显著降低（P﹤0.05）。缺氧条件下，转染PD-L1启动子于HepG2肝癌细胞，XAV939-PD-L1启动子组荧光素酶活性较未受到XAV939作用的PD-L1启动子组明显减低。提示，Wnt /β-catenin信号通路为PD-L1过表达的上游条件。染色质免疫共沉淀（Chip）显示，Wnt /β-catenin信号通路下游分子LEF1能够与PD-L1启动子基因片段结合，诱导PD-L1基因表达，进一步阐明了PD-L1过表达的相关分子机制。.动物实验中，利用常氧和缺氧肝癌细胞建立裸鼠肿瘤模型。缺氧组肿瘤负荷显著大于常氧组。PD-L1 抗体和XAV939可有效抑制肿瘤增殖。Western blotting结果显示，缺氧组肝癌细胞HIF-1α、PD-L1、Cyclin D1、VEGF、MMP9及Snail表达水平较常氧组显著升高；XAV939组肿瘤细胞PD-L1、Cyclin D1、VEGF、MMP9及Snail表达水平较对照组显著降低。.综上所述，本研究显示，缺氧肝癌细胞HIF-1α可通过激活Wnt /β-catenin信号通路上调PD-L1表达，促进肝癌细胞进展。本研究进一步揭示了缺氧调控 PD-L1 过表达促进肿瘤细胞进展的作用机制，阐明 HCC 缺氧对 TACE 疗效的影响，为临床上突破 TACE 治疗 HCC 的瓶颈提供理论基础和科学依据。