MicroRNA-30e* Suppresses Dengue Virus Replication by Promoting NF-κB–Dependent IFN Production

<div><p>MicroRNAs have been shown to contribute to a repertoire of host-pathogen interactions during viral infection. Our previous study demonstrated that microRNA-30e* (miR-30e*) directly targeted the IκBα 3′-UTR and disrupted the NF-κB/IκBα negative feedback loop, leading to hyperactivation of NF-κB. This current study investigated the possible role of miR-30e* in the regulation of innate immunity associated with dengue virus (DENV) infection. We found that DENV infection could induce miR-30e* expression in DENV-permissive cells, and such an overexpression of miR-30e* upregulated IFN-β and the downstream IFN-stimulated genes (ISGs) such as <i>OAS1</i>, <i>MxA</i> and <i>IFITM1</i>, and suppressed DENV replication. Furthermore, suppression of IκBα mediates the enhancing effect of miR-30e* on IFN-β-induced antiviral response. Collectively, our findings suggest a modulatory role of miR-30e* in DENV induced IFN-β signaling via the NF-κB-dependent pathway. Further investigation is needed to evaluate whether miR-30e* has an anti-DENV effect <i>in vivo</i>.</p></div

miR-30e* enhances expression of IFN-β.

<p>U937, HeLa and PBMC cells were transfected with 20 nM of miR-30e* mimics or negative control mimics (NC) and then infected with DENV2 at MOI of 1. (<b>A</b>) mRNA levels of IFN-β were measured by real-time RT-PCR. (<b>B</b>) Protein levels of IFN-β were determined by ELISA. Expression of IFN-induced genes <i>OAS1</i>, <i>MxA</i> and <i>IFITM1</i> in U937 cells (<b>C</b>) and HeLa cells (<b>D</b>) were determined by real-time RT-PCR at 24 h after transfection. Data show mean ± SD derived from three repeat experiments. **<i>p</i><0.01 (Student's t-test).</p

DENV infection induces miR-30e* expression.

<p>HeLa cells were infected with or without DENV1, 2 and 3 at MOI of 1 for 6 h. Expressions of miR-30e* was measured by qRT-PCR and normalized to the expression of U6 in each sample. Data show mean ± SD from three repeat experiments. **<i>p</i><0.01 (Student's t-test).</p

miR-30e* inhibition increases DENV2 replication.

<p>(<b>A</b>) U937 and HeLa cells were transfected with a specific miR-30e* inhibitor or negative control (Inhibitor NC) at a final concentration of 50 nM. The expression levels of miR-30e* were analyzed by real-time RT-PCR. (<b>B</b>) Prior transfection of U937 and HeLa cells with miR-30e* inhibitor accelerated DENV2 replication. DENV2 RNA levels were measured by real-time RT-PCR. Data show mean ± SD derived from three repeat experiments. **<i>p</i><0.01 (Student's t-test).</p

miR-30e* suppresses the replication of DENV2 in U937 and HeLa cells.

<p>U937, HeLa and PBMC cells were transfected with 20 nM of miR-30e* mimics or negative control mimics (NC) and then infected with DENV2 at MOI of 1. (<b>A</b>) The expression levels of miR-30e* were analyzed by real-time RT-PCR. The cellular viral RNA (<b>B</b>) and supernatant viral RNA (<b>C</b>) were assessed using real-time RT-PCR. Expression levels were normalized to <i>GAPDH</i>. Data show mean ± SD from three repeat experiments. **<i>p</i><0.01 (Student's t-test). (<b>D</b>) miR-30e*-transfected U937 and HeLa cells were infected with DENV2, and then the viral E, prM and Actin proteins were detected by immunoblotting analysis. (<b>E</b>) miR-30e*-transfected HeLa cells were infected with DENV2, and then stained with anti-E antibody as well as DAPI, and subsequently a secondary antibody conjugated to Rhodamine was used to visualize stained E proteins. Samples were inspected by fluorescence microscope at a magnification of 200×.</p