MiR-4334-5p Facilitates Foot and Mouth Disease Virus Propagation by Suppressing Interferon Pathways via Direct Targeting ID1

Figures

• 

  Figure 1. MiR-4334-5p expression was induced by FMDV. (A,B). Porcine PK-15 cells were challenged with FMDV at 1 MOI and 0.1 MOI. Cells were harvested at indicated time points to examine the expression of miR-4334-5p by qRT-PCR, and miR-16 was detected as an internal control. The data shown represent of three independent experiments with similar results, and normalized to miR-16; Error bars is standard deviation (SD). Significance was calculated by Student’s t-test, *** p < 0.001; ** p < 0.01; * p < 0.05.

• 

  Figure 2. FMDV replication was up-regulated by miR-4334-5p mimics. (A). MiR-4334-5p mimics or scramble mimics (NC) were transfected into PK-15 cells for 18 h or 24 h, respectively. Cells were collected and then quantified miR-4334-5p expression by qRT-PCR, and miR-16 was examined as an internal control in parallel. Data shown are means ± SD from triplicate assays, ** p < 0.01. (B). MiR-4334-5p mimics or scramble mimics (NC) were transfected into PK-15 cells for 24 h, cells were challenged with FMDV at 0.1 MOI for 6 h or 8 h, and then cells were harvested to quantify VP1 expression by qRT-PCR, and the expression level was normalized to β-actin. The data are means ± SD from triplicate assays, *** p < 0.001; ** p < 0.01 (C). PK-15 cells were treated as in (B), the cells were lysed and then subjected to Western blot, VP1 and β-actin antibodies were used to detect the protein expression. (D). PK-15 cells were treated as in (B), after infected with the indicated time, the supernatants were collected to measure the virus titers by TCID50 (Median Tissue Culture Infectious Dose) assay. Results shown are means ± SD from triplicate assays, * p < 0.05.

• 

  Figure 3. FMDV replication was down-regulated by miR-4334-5p inhibitors. (A). MiR-4334-5p inhibitors or scramble control (NC) were transfected into PK-15 cells for 24 h independently. Cells were harvested and quantified miR-4334-5p expression by qRT-PCR, and miR-16 was examined as an internal control in parallel. Data shown are means ± SD from triplicate assays, ***, p < 0.001. (B). MiR-4334-5p inhibitors or scramble control (NC) were transfected into PK-15 cells for 24 h, cells were challenged with FMDV at 0.1 MOI for 8 h, and then cells were harvested to quantify VP1 expression by qRT-PCR, and the expression level was normalized to β-actin. The data are means ± SD from triplicate assays, **, p < 0.01 (C). PK-15 cells were treated as in (B), the cells were lysed and then subjected to Western blot, VP1 and β-actin antibodies were used to detect the protein expression. (D). PK-15 cells were treated as in (B), after infected with the indicated time, the supernatants were collected to measure the virus titers by TCID50 (Median Tissue Culture Infectious Dose) assay. Results shown are means ± SD from triplicate assays, *, p < 0.05.

• 

  Figure 4. miR-4334-5p directly targets the 3′-UTR of ID1 gene. (A). qRT-PCR analysis of the effect of miR-4334-5p on ID1 mRNA expression in PK-15 cells transfected with miR-4334-5p mimics or scramble mimics (NC) at 24 h post transfection, normalized to β-actin. The results shown are means ± SD from triplicate assays, **, p < 0.01. (B) Western blot analysis of miR-4334-5p on ID1 expression in PK-15 cells transfected with miR-4334-5p mimics or scramble mimics (NC) at 24 h post transfection, and β-actin was used as a control to indicate the even loading (C). Bioinformatic prediction of interaction between miR-4334-5p and the 3′-UTR of swine ID1 using RNA22 and BiBiServ2. For each schematic, the upper sequence is mature miR-4335-5p, the middle sequence is the binding site of miR-4334-5p in 3ʹ-UTR of swine ID1, and the lower sequence is the mutated sequence of 3′-UTR in ID1 gene. The seed sequence is underlined. (D). Schematic overview of the putative binding sites or mutation of miR-4334-5p binding-sites in 3′-UTR of ID1 mRNA. (E). The ID1 luciferase reporter plasmid was co-transfected with various amount miR-4334-5p mimics or scramble mimics (NC) into BHK-21 cells. Data are represented of…

• 

  Figure 5. The interferon and antiviral genes expression were suppressed by miR-4334-5p via targeting ID1. (A,B). MiR-4334-5p mimics or scramble mimics (NC) were transfected into PK-15 cells for 24 h, respectively. Cell were infected with FMDV at 0.1 MOI for another 8 h. And then cells were harvested to detect the IFN-β expression level by qRT-PCR and IFN-β ELISA kit. Results shown are representative of three independent experiments, with each performed in triplicates. Bars represent SD of the mean. Asterisks indicate significance, according to Student’s t-test, ***, p < 0.001; **, p < 0.01, *, p < 0.05. (C–E). PK-15 cells were treated in the same way as in (A,B). Cells were harvested to quantify TNFα, OAS and ISG expression by qRT-PCR, β-actin was determined as an internal control. Results shown are means ± SD from triplicate assays. ***, p < 0.001; **, p < 0.01; *, p < 0.05.