miR‐127‐5p negatively regulates enterovirus 71 replication by directly targeting SCARB2

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  Figure 1. miR‐127‐5p inhibits SCARB2 expression by targeting its 3′ UTR. (A) HeLa and HepG2 cells were stained with the anti‐SCARB2 antibody (solid lines) or normal mouse IgG (dotted lines), and then the surface SCARB2 expression was analyzed by flow cytometry. (B) Susceptibility of HeLa and HepG2 cells to EV71. CPE of HeLa and HepG2 cells infected with EV71. HeLa and HepG2 cells were infected with EV71 at an MOI of 0.1. At 48 h postinfection, these cells were imaged via light microscopy. Scale bar, 100 μm. (C) The cells were lysed and total RNA was extracted. Level of miR‐127‐5p was determined by quantitative real‐time PCR analysis. (D and E) HeLa and HepG2 cells were transfected with miR‐127‐5p mimics or NC mimics (final concentration, 50 nm) for 48 h. (D) The cells were lysed and total RNA was extracted. SCARB2 mRNA level was determined by quantitative real‐time PCR analysis. (E) SCARB2 protein expression in HeLa and HepG2 cells was detected by western blot analysis. The numbers represent the relative density of the band in comparison to the corresponding control normalized to GAPDH. Value of NC mimics treatment is set at 1.00 (100%). (F and G) HeLa and HepG2 cells were transfected with…

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  Figure 2. EV71 infection upregulates miR‐127‐5p expression. (A) HeLa and (B) HepG2 cells were infected with EV71 at an MOI of 10. The cells were harvested at 0 h, 12 h, and 24 h postinfection. Total cellular RNA was extracted and the level of miR‐127‐5p was determined by quantitative real‐time PCR. (C) HeLa and (D) HepG2 cells were infected with EV71 at an MOI of 10. The cells were lysed, and mRNA level and protein expression of SCARB2 were determined at the indicated times. The numbers represent the relative density of the bands normalized to the control. Data are representative of at least three independent experiments, with each measurement performed in triplicate (mean ± SD of fold‐change). *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3. EV71 gene expression and replication are attenuated by miR‐127‐5p overexpression. (A) Flow cytometric analysis was performed to assess the surface SCARB2 level of HeLa and HepG2 cells after 48 h transfection. The cells transfected with NC mimics (final concentration, 50 nm) were stained with the anti‐SCARB2 antibody (solid lines), and transfected with miR‐127‐5p mimics (final concentration, 50 nm) were stained with anti‐SCARB2 antibody (gray region), or the cells were stained with normal mouse IgG (dotted lines, the same result for the cells treating with NC mimics or miR‐127‐5p mimics). (B) HeLa and HepG2 cells were transfected with miR‐127‐5p mimics or NC mimics (final concentration, 50 nm) for 48 h and then infected with EV71 at an MOI of 2.0. The cells were harvested at 12 h and 24 h postinfection, and western blot analysis of the expression levels of EV71 VP1 was performed. The numbers below each band denote the relative density of the bands normalized to the control. (C and D) HeLa cells were transfected with miR‐127‐5p mimics or NC mimics (final concentration, 50 nm) for 48 h and then infected with EV71 (MOI = 2) at the indicated times. (C) SCARB2 mRNA and (D) EV71 VP1…

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  Figure 4. Suppression of endogenous miR‐127‐5p enhances EV71 replication. (A) Flow cytometric analysis was used to assess the surface SCARB2 level of HeLa and HepG2 cells after 48 h transfection. The cells transfected with NC inhibitor (final concentration, 100 nm) were stained with the anti‐SCARB2 antibody (solid lines), and transfected with miR‐127‐5p inhibitor (final concentration, 100 nm) were stained with anti‐SCARB2 antibody (gray region), or the cells were stained with normal mouse IgG (dotted lines, the same result for the cells treating with NC inhibitor or miR‐127‐5p inhibitor). (B) HeLa and HepG2 cells were transfected with miR‐127‐5p inhibitor or NC inhibitor (final concentration, 100 nm) for 48 h and then infected with EV71 at an MOI of 2.0. The cells were harvested at 12 h and 24 h postinfection, and western blot analysis of the expression levels of EV71 VP1 was performed. The numbers below each band denote the relative density of the bands normalized to the control. (C–E) HeLa cells were transfected with miR‐127‐5p inhibitor or NC inhibitor for 48 h and then infected with EV71 (MOI = 2.0) at the indicated times. (C) SCARB2 mRNA and (D) EV71 VP1 mRNA levels were determined by…

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  Figure 5. miR‐127‐5p does not influence intracellular replication of EV71. (A) HeLa cells were transfected with miR‐127‐5p mimics or NC mimics (final concentration, 50 nm) for 48 h and the cells were infected by EV71 at an MOI of 10 in PBS containing 0.05% EDTA for 1 h at 4 °C. The cells were washed, lysed and EV71 VP1 protein level was determined by western blot and normalized to GAPDH. The numbers denote the relative density of the bands normalized to the control. (B–D) L929 cells were cotransfected with EV71 genomic RNA and miR‐127‐5p or NC mimics for 12 h and 24 h. Levels of miR‐127‐5p (B) and EV71 RNA (C) were determined by quantitative real‐time PCR analysis. (D) EV71 VP1 level was determined by western blot and normalized to GAPDH. The numbers denote the relative density of the bands normalized to the control. Data are representative of at least three independent experiments, with each measurement performed in triplicate (mean ± SD of fold‐change). ***P < 0.001.