Contradicting roles of miR-182 in both NK cells and their host target hepatocytes in HCV
Contradicting roles of miR-182 in both NK cells and their host target hepatocytes in HCV
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DOI：
10.1016/j.imlet.2015.10.013
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发表时间：
2016-01-01
影响因子：
4.4
通讯作者：
Abdelaziz, Ahmed I.
中科院分区：
文献类型：
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作者：
El Sobky, Shereen A.;El-Ekiaby, Nada M.;Abdelaziz, Ahmed I.
背景和目标：自然杀伤细胞是参与病毒根除的先天免疫的一部分，并被证明受到HCV感染的极大影响。通过microRNA对NK细胞功能的表观遗传调节以前没有被有效地研究过，并且从未在HCV感染中进行过研究;因此，本研究的目的是首次评估microRNA在调节HCV感染患者的NK细胞功能中的作用，从而在靶HCV感染的Huh 7细胞中进行病毒复制。从HCV感染的患者以及对照的PBMC中分离NK细胞，并使用HCV感染的肝活检以及病毒感染的Huh 7细胞。对于Huh 7细胞的感染，首先将病毒载体在体外转录成病毒RNA，然后将其用于感染na？ve Huh 7细胞。然后收集来自感染细胞的上清液并用于进一步感染。为了操作NK细胞或Huh 7细胞，通过脂质转染方法转染miR-182模拟物和抑制剂。从每个细胞群中提取RNA，进行逆转录。使用qPCR定量基因表达和病毒载量。结果：患者和对照组之间的NKG 2A和NKG 2D筛查显示NKG 2A的表达没有差异，而NKG 2D的表达被发现下调。有鉴于此，进行了生物信息学分析，结果显示miR-182在抑制性受体NKG 2A和活化性受体NKG 2D上、其配体ULBP 2上以及病毒基因组本身上都具有潜在的结合位点。在HCV感染患者的NK细胞中，发现miR-182与对照组相比过度表达;发现其异位表达降低NKG 2D mRNA水平，而与未转染细胞相比，miR-182抑制剂能够降低NKG 2A mRNA水平。此外，共培养基因型4或2 HCV感染的Huh 7细胞与HCV感染患者的miR-182模拟NK细胞显示病毒复制减少，表明NK细胞功能增强。另一方面，发现miR-182和ULBP 2在HCV肝组织和HCV感染的Huh 7细胞中与其对照相比均下调。发现与对照相比，miR-182模拟物在基因型4和2 HCV感染的靶细胞（Huh 7）中降低ULBP 2 mRNA并增加病毒复制，而miR-182抑制剂在细胞模型中降低病毒复制。以前从未研究过miR-182，无论是在HCV感染中还是在NK细胞中，我们发现与对照组相比，它在HCV感染患者的肝组织和NK细胞中都有失调的表达。除此之外，发现miR-182在效应细胞和其HCV感染的靶细胞中对HCV复制具有矛盾的作用。(C)2015年由Elsevier B. V.代表欧洲免疫学会联合会出版。
Background and aim: Natural killer cells are part of the innate immunity involved in viral eradication and were shown to be greatly affected by HCV infection. Epigenetic regulation of NK cell function by microRNAs was not efficiently studied before and was never studied in HCV infection; therefore the aim of this study was to assess for the first time the role of microRNAs in regulating the function of NK cells of HCV-infected patients and hence viral replication in the target HCV-infected Huh7 cells.Methodology: NK cells were isolated from PBMCs of HCV-infected patients as well as controls, and HCV-infected liver biopsies as well as Huh7 cells infected with the virus were used. For the infection of Huh7 cells, first viral vector was in-vitro transcribed into viral RNA that was then used to infect na?ve Huh7 cells. Supernatant from the infected cells was then collected and used for further infection. For manipulation of NK cells or Huh7 cells, miR-182 mimics and inhibitors were transfected via lipofection method. RNA was extracted from each cell population, reverse transcribed. Gene expression as well as viral load was quantified using qPCR.Results: Screening of NKG2A and NKG2D between patients and controls showed no difference in expression of NKG2A, while NKG2D was found to be downregulated. In view of that, bioinformatics analysis was performed and showed that miR-182 has potential binding sites on both the inhibitory receptor NKG2A and the activating receptor NKG2D, and on its ligand ULBP2, as well as on the viral genome itself. In NK cells of HCV-infected patients, miR-182 was found to be over-expressed compared to controls; its ectopic expression was found to decrease NKG2D mRNA level, while miR-182 inhibitors were able to decrease NKG2A mRNA compared to untransfected cells. In addition, co-culturing genotype 4 or 2 HCV-infected Huh7 cells with miR-182 mimicked NK cells of HCV-infected patients showed decreased viral replication, suggesting an enhanced NK cell function. On the other hand, miR-182 and ULBP2 were both found to be downregulated in HCV liver tissues and HCV-infected Huh7 cells compared to their controls. miR-182 mimics were found to decrease ULBP2 mRNA and increase viral replication in genotypes 4 and 2 HCV-infected target (Huh7) cells compared to controls, while miR-182 inhibitor decreased viral replication in the cell models.Conclusion: miR-182 was never investigated before, neither in HCV infection nor in NK cells, and we found it to have dysregulated expression in both liver tissues and NK cells of HCV-infected patients compared to control. In addition to that, miR-182 was found to have a contradicting effect in both effector cell and its HCV-infected target cell regarding HCV replication. (C) 2015 Published by Elsevier B.V. on behalf of European Federation of Immunological Societies.