Enterovirus 71 Suppresses miR-17-92 Cluster Through Up-Regulating Methylation of the miRNA Promoter

Figures

• 

  FIGURE 1. EV71 infection downregulated miR-17-92 cluster. (A) The heatmap showed the expression level of members of miR-17-92 cluster in mock- and EV71-infected HT-29 cells by small RNA deep sequencing. (B) Expression of mature miRNAs of the miR-17-92 cluster was analyzed in EV71-infected HT-29 cells at indicated time points by real-time quantitative PCR, using U6 rRNA as an internal control. Values were mean of triplicate experiments and presented in relative to the level of the mock infection. Mock infection was assigned with a value of 1. The data were presented as mean ± SEM (n = 3, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (C) RT-PCR analysis of the pri-miR-17-92 cluster expression in EV71-infected cells at indicated time points normalized against GAPDH transcript levels.

• 

  FIGURE 2. The expression level of miR-17-92 cluster affected EV71 replication. (A) RT-PCR analysis of pri-miR-17-92 cluster expression after HT-29 or Caco-2 cells were transfected with si-pri-mir-17-92 for 48 h. (B, C) HT-29 cells were infected with EV71 (MOI = 1) prior to being transfected with si-pri-mir-17-92 for 24 h. EV71 VP1 expression level was determined via In-cell Western and normalized by DRAQ5 (B), and viral RNA level was quantified via real-time quantitative PCR at indicated time points normalized against GAPDH transcript level (C). (D) Quantitative real-time-PCR analysis of miR-17-92 component expression in HT-29 cells when the cells were transfected with MSCV-miR-17-92 vector or negative control (MSCV-n.c) for 48 h. (E) Titers of progeny EV71 in the culture supernatant of infected-cells before the cells were transfected with MSCV-miR-17-92 vector or negative control. All experiments were performed three times and the representative results were shown. The data were presented as mean ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

• 

  FIGURE 3. miR-17-5p, miR-19a, and miR-19b directly targeted the EV71 genome. (A) Potential target sites of mature miRNAs of the miR-17-92 cluster in the EV71 genome. Computational analysis with RNA-hybrid software yielded potential binding sites for miR-17, miR-20a, and miR-19a/b in the EV71 genome. (B) EV71 genome containing presumptive miR-17-92 target sequences, was cloned into the multiple cloning sites at the 3′ of the firefly luciferase reporter gene (luc2) in the luciferase expression plasmid (pmirGLO). Either control plasmid (pmirGLO-control), pmirGLO-viral genome WT (pmirGLO-VG WT) or pmirGLO-viral genome mut (pmirGLO-VG-m) was co-transfected with individual miRNA mimic in HT-29 cells. The luciferase activity of pmirGLO-VG WT group was repressed by transfecting miR-17 and miR-19a/b while mutation of a predicted miRNAs binding sites abolished miRNAs-mediated repression. (C,D) HT-29 cells and Caco-2 were infected with EV71 (MOI = 1) after being transfected with miR-17 or miR-19b mimic for 24 h. EV71 VP1 expression level was determined via In-cell Western and normalized by DRAQ5 (C) or Western blot (D). The symbols “+” and “-” indicate treatment and no treatment, respectively, by the…

• 

  FIGURE 4. Altered DNA methylation of the CpG island in the miR-17-92 cluster promoter in EV71 infection cells. (A) DNA was isolated from mock and EV71-infected cells (n = 3). Data were presented as the average percent of unmethylated or methylated DNA of the miR-17-92 promoter. Statistical difference was determined by comparing the % unmethylated with % methylated for each group using QIAampDNA Mini Kit. (B) Western analysis of the expression levels of DNA methyltransferase (DNMT) enzymes 1, 3A, and 3B in EV71-infected HT-29 cells at the indicated time point. (C) HT-29 cells were transfected with DNMT3A- or 3B-specific siRNA for 48h and then detected by Western blot. The numbers represent the relative density of the band in comparison to the corresponding negative control normalized to GAPDH. Value of siRNA-n.c treatment is set at 1.00 (100%). (D) HT-29 cells were transfected with DNMT3A- or 3B-specific siRNA for 24 h and infected with EV71 at an MOI of 1. DNA was isolated and methylated DNA of the miR-17-92 promoter was determined by QIAampDNA Mini Kit. Shown is the percentage of DNA methylation, n = 4. (E) Pri-miR-17-92 cluster expression in cells transfected with si-DNMT3A- or 3B for 24…

• 

  FIGURE 5. miR-17-92 was reduced in serum of EV71-infected patients. (A–F) The expression levels of mature miR-17-92 in serum specimens of EV71-infected patients by real-time quantitative PCR, and their correlation with disease severity. The EV71-infected patients were divided into a mildly ill group (sample = 25) and a severely ill group (sample = 22) as based on criteria of the Handbook for Treatment of HFMD.