MicroRNA screening identifies miR-134 as a regulator of poliovirus and enterovirus 71 infection

Figures

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  Figure 1. miR-134 inhibits sabin-2 replication in HEp-2c and Caco-2 cells. (a) Schematic of the experimental design. To evaluate if antiviral effect of miR-134 is cell type-specific, HEp-2c (b) and Caco-2 (c) cells were reverse-transfected with miR-134-5p mimic or miR mimic non-targeting scrambled negative control (miR-NTC) at a final concentration of 25 nM. After 48 h, the transfected cells were infected with Sabin-2 at MOI of 0.01 for 24 h and the virus titers were determined by plaque assay. Error bars represent s.e.m. and the significant differences are compared with miR-control. Each experiment was performed in quadruplicate and each experiment was repeated twice. The results were reproduced and the data in the figure were representative of 8 plates. *P<0.05, **P<0.01.

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  Figure 2. RAN is a target of miR-134. (a) Sequence of the putative binding site between miR-134 and 3′UTR of RAN. (b) and (c) Hep-2c cells were reverse-transfected with miR-134-5p or miR mimic non-targeting scrambled negative control (miR-NTC) at a final concentration of 25 nM. After 48 h, total RNA was isolated from HEp-2c cells, and then miR-134 (b) and RAN (c) mRNA levels were determined by qRT-PCR. The data were normalized to the 18S rRNA gene and were expressed as fold change relative to mock-transfected cells. The significant differences are compared with miR-control. Each experiment was performed in quadruplicate and each experiment was repeated twice. The results were reproduced and the data in the figure were representative of 8 plates. Error bars represent s.e.m. *P<0.05, ***P<0.001.

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  Figure 3. Silencing of RAN inhibits PV replication in HEp-2c and Caco-2 cells. The cells were reverse-transfected with siRAN or siRNA control at a final concentration of 25 nM. After 48 h, (a) total RNA was isolated from HEp-2c cells and RAN mRNA level was determined by qRT-PCR. The data were normalized to the 18S rRNA gene and were expressed as fold change relative to siRNA control. The transfected Hep-2c (b) and Caco-2 (c) cells were infected with Sabin-2 strain at an MOI of 0.01 for 24 h, and then the virus titers were determined by plaque assay. The significant differences are compared with siRNA control. Error bars represent s.e.m. of quadruplicate. Each experiment was performed in quadruplicate and each experiment was repeated twice. The results were reproduced and the data in the figure were representative of 8 plates. **P<0.01, ***P<0.001.

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  Figure 4. miR-134 targets EV71 replication in RD cells. (a) Schematic of the experimental design. (b) RD cells were reverse-transfected with miR-134-5p or miR mimic non-targeting scrambled negative control (miR-NTC) at final concentration of 25 nM. 48 h later the cells were infected with EV71 at MOI of 0.01 for 24 h and the antiviral effect of miR-134 was determined by plaque assay. Error bars represent s.e.m. of of quadruplicate. Each experiment was performed in quadruplicate and each experiment was repeated twice. The results were reproduced and the data in the figure were representative of 8 plates. The significant differences are compared with miR-control. ***P<0.001.

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  Figure 5. miRNAs inhibit sabin-2 replication in HEp-2c cells. Cells were reverse-transfected with miRNA mimic or miR-control mimic (miR-control) at a final concentration of 25 nM. After 48 h, the transfected cells were infected with Sabin-2 at MOI of 0.01 for 24 h and the virus titers were determined by ESLIA assay.

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  Figure 6. (a) optimization of transfection using miRIDIAN microRNA mimic housekeeping positive control #2 (GAPDH) (GE Dharmacon; Cat # CP-001000-02). (b) Optimization of positive control (siPol) for virus replication (CDC, provided by Dr Steve Oberste).