miR-181a通过SIRT1参与HCV感染慢性化的作用机制

Infection of hepatitis C virus (HCV) can cause decrease of adaptive immunity of host and cell senescence, which leads to the decline of clearance of HCV and persistent viral infection. According to our previous studies, the CD4+T cells of untreated HCV infected patients are aging, the immune function of CD4+T cells is decreased, which may be relate with DUSP6 regulated by miR-181a. Recently, we found the senescence of T cell in chronic HCV infection maybe anti-regulated by miR-181a-SIRT1 pathway. We speculated that after HCV infection, miR-181a may bidirectional regulate T cell function, contributing virus persistence, and be affected by other immune factors. To verify our hypothesis, we plan to collect T cells of chronically HCV-infected individuals (prior or after antiviral treatment) and healthy subjects, compare the relationship of miR-181a and SIRT1 expressions in T cells with the HCV load and the liver functional tests. We will also evaluate the effect of SIRT1 on CD8+T cells by assaying the cytotoxic function of T cells against HCVcc infected Huh7.5 cells. The goal of this proect is to illuminate the mechanism of miR-181a-SIRT1 pathway in regulating HCV chronic infection. The outcomes of this project could provide evidence for miR-181a-SIRT1 pathway as a potential immunol therapeutic target for chronic HCV infection.

HCV感染损害适应性免疫，导致细胞衰老，进而病毒持续性感染。我们前期研究证明：HCV慢性感染者CD4+T细胞免疫功能下降可能与miR-181a调节DUSP6活性，导致T细胞功能耗竭和衰老有关。近期实验表明，HCV诱导的T细胞衰老可被miR181a-Sirt1通路对抗调节。因此，我们认为HCV感染后，miR-181a调节T细胞的功能可能是双向，并受其它免疫分子所影响。依此提出本研究思路：收集HCV慢性感染者和HCV治愈者以及健康对照者的外周血T细胞，检测miR-181a及SIRT1的表达水平并分析其与HCV病毒载量、肝功能的相关性。体外抑制miR-181a或SIRT1活性后，对比CD8+T细胞对HCVcc感染的Huh7.5肝细胞的杀伤力变化。明确miR-181a通过SIRT1调节HCV感染慢性化的作用机制，为研究miR-181a-SIRT1通路阻止丙型肝炎慢性化的免疫治疗提供依据。

HCV感染损害适应性免疫，导致细胞衰老，进而病毒持续性感染。我们前期研究证明：HCV.慢性感染者CD4+细胞免疫功能下降可能与miR-181a调节DUSP6活性，导致T细胞功能耗竭和衰老有关。近期实验结果表明，HCV诱导的T细胞衰老可被miR181a-Sirt1通路对抗调节。因此，我们认为HCV感染后，miR-181a调节CD4+细胞T的功能可能是双向，并受其它免疫分子所影响。.mir-181a 和SIRT1 双荧光素酶实验显示hsa-mir-181a-5p显著下调h-SIRT1-3UTR的luciferase的表达，说明两者间存在直接相互作用。HCV诱导的CD4+T细胞衰老，可被ΔNp63-miR181a-Sirt1通路对抗调节。和年龄匹配的健康对照者相比，慢性HCV感染者CD4+T细胞更衰老。实验揭示转录因子ΔNp63的升高能导致miR-181a的下降，进而过表达抗衰老分子SIRT1。在HCV患者CD4+T细胞中沉默ΔNp63或SIRT1以及重构miR-181a的表达能加速T细胞衰老，表现为端粒酶活性下降，端粒长度缩短，增加衰老相关的β半乳糖苷酶 (SA-β-gal)表达，以及与增殖相关的EdU掺入下降。.与健康对照组相比，HCV组和HCV治愈组CD8+T细胞上SIRT1、PD-1、TIM-3表达上升，p53、p21水平下降，HCV组和HCV治愈组无统计学差异。与健康对照组相比，HCV组外周血的IL-6和TNF-α升高，HCV治愈组IL-6仍比健康对照组高，而TNF-α下降至与健康对照组无统计学差异。提示HCV 感染者CD8+T 细胞免疫衰老，经直接抗病毒药物（Direct-acting antiviral agents，DAA）治疗后免疫衰老缓解，CD8+T细胞功能部分恢复。.miR-181a通过SIRT调节HCV感染慢性化的作用机制，为研究miR-181a-SIRT1通路阻止丙型肝炎慢性化的免疫治疗提供依据。