Contradicting roles of miR-182 in both NK cells and their host target hepatocytes in HCV.

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 naїve 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.