MicroRNA 876-5p modulates EV-A71 replication through downregulation of host antiviral factors

Figures

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  Fig. 1. miR-876-5p facilitated the EV-A71 infection cycle in human neuroblastoma SF268 cells. a SF268 cells were infected with EV-A71 at an MOI of 1, and the cells were harvested at different time points after infection, as indicated. The RNA was the isolation and expression level of miR-876-5p, which was determined through real-time quantitative polymerase chain reaction (RT-qPCR); b The expression level of miR-876-5p was detected through RT-qPCR after in the pmiR-876-5p transfected cells; c SF268 cells were expressed with miR-876-5p (lanes 2–3), followed by infection with EV-A71. The intracellular EV-A71 viral proteins, 3D/3CD, and VP1–3 were detected through Western blotting (lane 1: control cells; lanes 2–3: miR-876-5p overexpressed; lane 4: mock-infected cells). β-actin was loaded as the internal control. All experiments were repeated three times. ** P < .01

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  Fig. 2. miR-876-5p preferentially regulated the EV-A71 life cycle. a SF268 cells were introduced to an anti-miR-876-5p inhibitor and then cultivated for 24 h. The expression level of miR-876-5p was quantified by real-time polymerase chain reaction (RT-qPCR). The anti-miR-876-5p-inhibitor-treated cells were infected with EV-A71 (MOI = 1) for 24 h. Both the (+)strand of viral RNA (b) and (−)strand of viral RNA (c) were quantified through RT-qPCR; d protein expression levels of viral 3D/3CD and VP1–3 were detected by Western blot using anti-3D and anti-VP specific antibodies (lane 2: Negative control cells; lane 3: anti-miR-876-5p inhibitor treatment); e The infectivity of EV-A71 progeny from the anti-miR-876-5p-inhibitor-treated cells was assessed in the plaque assay. The virus titers are shown in PFU/mL. The data shown are based on the mean of three independent experiments. * P < .05; ** P < .01

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  Fig. 3. CREB5 was a direct-targeted gene of miR-876-5p. a Predicted binding sites between miR-876-5p and 3’UTR of CREB5. b Luciferase activities of SF268 cells were determined through the luciferase reporter assay by cotransfection with CREB5–3’UTR or CREB5–3’UTR-mutant and miR-876-5p or miR-control. The expression level of CREB5 at mRNA (c) and protein (d) levels was measured through real-time quantitative polymerase chain reaction and Western blotting in SF268 cells transfected with miR-876-5p (lane 2), anti-miR-876-5p inhibitor (20 and 40 nM; lanes 3 and 4), or corresponding controls (lane 1). All experiments were repeated three times. * P < .05; ** P < .01

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  Fig. 4. CREB5 repressed EV-A71 viral replication and expression. a SF268 cells were mock-infected or infected with EV-A71 (MOI = 1) for different time points as indicated (lanes 2–5. 12–48 h after infection). Cell lysates were then harvested for detection of CREB5 protein levels by Western blotting using the anti-CREB5 specific antibody. b Mice were treated with the miRNA hairpin inhibitor negative control in phosphate buffered saline (lane 1) or anti-miR-876-5p inhibitor for 24 h and infected with EV-A71. The protein extracted from the site of injection in the abdominal muscle was collected for CREB5 and viral protein investigations; VP1–3 analysis was performed with Western blotting using specific antibodies as indicated. Lane 1, the miRNA hairpin negative control and no EV-A71 infection; lane 2, the miRNA negative control of mice infected with EV-A71 or anti-miR876-5p inhibitor-treated (lane 3) mice. c Quantitative polymerase chain reaction (qPCR) analysis of CREB5 mRNA level in si-CREB5-transfected or CREB5-overexpressed cells. d Expression of CREB5 determined by Western blotting in pCMV-CREB5-Flag-transfected cells (lane 2) or siCREB5-transfected cells (lanes 3–4). Lane 1, cells…

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  Fig. 5. Downregulation of miR-876-5p inhibited EV-A71 replication and expression by modulating CREB5. SF268 cells were transfected with either anti-miR-876-5p-inhibitor only or in combination with si-CREB5 or si-Scramble as negative control. a SF268 cells were treated with the miRNA hairpin negative control (anti-miR-control), anti-miR-876-5p, anti-miR-876-5p and si-scramble RNA (the negative control for si-CREB5 RNA), or anti-miR-876-5p and si-CREB5 RNA for 24 h, followed by EV-A71 infection. The cells were harvested at 24 h after the EV-A71 infection, and viral RNA was examined by real-time-qPCR of each sample. b EV-A71 viral 3D protein was detected by Western blotting using anti-3D specific antibody. β-actin was detected as the loading control. c Progeny viruses were measured in culture supernatant by a plaque-forming assay. All experiments were repeated three times. * P < .05; ** P < .01