Department of Internal Medicine, Saga Medical School, Saga University, Saga 849‑8501, Japan.
Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido 060‑8638, Japan.
Int J Mol Med. 2018 Aug;42(2):957-965. doi: 10.3892/ijmm.2018.3693. Epub 2018 May 18.
Although interferon (IFN)‑based treatment of patients with chronic hepatitis C virus (HCV) infection is widely applied, treatment resistance is often observed in patients with advanced liver fibrosis. Given that the molecular mechanisms of IFN resistance in liver fibrosis remain elusive, the present study investigated the effects of extracellular matrix (ECM) on IFN signaling in hepatic cells. The native HuH‑7 human hepatoma cell line and HuH‑7 cells were stably transfected with full‑length HCV‑RNA fused with Renilla luciferase (OR6 cells) were cultured on ECM‑coated dishes or non‑coated plastic dishes (NDs), and treated with human IFN‑α. In Huh‑7 cells cultured on coated dishes, the IFN‑stimulated response element (ISRE) luciferase activity was measured following ISRE plasmid transfection and the expression of IFN‑stimulated genes (ISG) were significantly lower than those in cells cultured on NDs. In addition, after IFN‑α treatment, the amount of HCV‑RNA and viral protein produced by OR6 cells cultured on coated dishes was higher than that produced by cells cultured on NDs. When cells were treated with β1‑integrin‑blocking antibody to disrupt the cell‑matrix interaction, the ISRE luciferase activity was restored, and the protein expression of ISG was increased, while that of HCV proteins was suppressed. Treatment of cells with integrin‑linked kinase (ILK) inhibitor or focal adhesion kinase (FAK) inhibitor restored the ISRE luciferase activity and expression of ISG proteins. These results suggested that β1‑integrin‑mediated signals affected the IFN signaling and promoted HCV replication. Therefore, the accumulation of ECM in liver fibrosis may impair IFN signaling through β1‑integrin‑mediated signaling involving ILK and FAK.
虽然基于干扰素 (IFN) 的慢性丙型肝炎病毒 (HCV) 感染患者治疗方法已被广泛应用,但在晚期肝纤维化患者中常观察到治疗耐药性。鉴于肝纤维化中 IFN 耐药的分子机制仍不清楚,本研究探讨了细胞外基质 (ECM) 对肝实质细胞中 IFN 信号的影响。本研究使用天然 HuH-7 人肝癌细胞系和全长 HCV-RNA 与 Renilla 荧光素酶融合的 HuH-7 细胞(OR6 细胞)进行稳定转染,在 ECM 涂层培养皿或非涂层塑料培养皿(NDs)上培养,并用人 IFN-α处理。在包被培养皿中培养的 Huh-7 细胞中,在转染 ISRE 质粒后测量 IFN 刺激反应元件 (ISRE) 荧光素酶活性,细胞培养在 NDs 上时 IFN 刺激基因 (ISG) 的表达明显较低。此外,在用 IFN-α处理后,OR6 细胞在包被培养皿中培养时产生的 HCV-RNA 和病毒蛋白的量高于在 NDs 中培养时产生的量。当用β1-整联蛋白阻断抗体处理细胞以破坏细胞-基质相互作用时,ISRE 荧光素酶活性恢复,ISG 蛋白的表达增加,而 HCV 蛋白的表达受到抑制。用整合素连接激酶 (ILK) 抑制剂或粘着斑激酶 (FAK) 抑制剂处理细胞可恢复 ISRE 荧光素酶活性和 ISG 蛋白的表达。这些结果表明,β1-整联蛋白介导的信号影响 IFN 信号转导并促进 HCV 复制。因此,肝纤维化中 ECM 的积累可能通过β1-整联蛋白介导的信号转导,涉及 ILK 和 FAK,损害 IFN 信号转导。