Figure 1.
Knockdown of DGCR8 using DsiRNA.
(A) Relative expression of DGCR8 gene measured by qPCR verifying knockdown. (n = 3, **** = p values of <0.0001) (B) Transfection Control using fluorescent-labeled transfection control duplex TYE 563 DS to monitor transfection efficiency.
Figure 2.
miRNA depleted HT29 cells is not able to support EV71 replication.
DGCR8 knockdown HT29 cells and control HT29 cells (scrambled DsiRNA) were infected with EV71 (MOI of 1). Total intracellular RNA were harvested at 36 hpi, converted to cDNA and measured by qPCR with primers specific to viral VP1. Total intracellular protein were harvested at at 36 hpi and measured by western blot (A) Relative expression of EV71 viral RNA measured by qPCR. (n = 3, **** = p values of <0.0001) (B) Western blot analysis for EV71 VP1 viral protein. (C) Cell viability assessed at 72 hpi using vital dye trypan blue. (n = 3, ** = p values of <0.01).
Figure 3.
EV71 is unable to establish infection in both RKO and RD miRNAs depleted cells.
DGCR8 knockdown cells and control cells (scrambled DsiRNA) in both RKO and RD cell line were infected with EV71 (MOI of 1). Total intracellular RNA were harvested at 12 hpi, converted to cDNA and measured by qPCR with primers specific to viral VP1. (A) Relative expression of DGCR8 gene in miRNAs depleted RKO cells measured by qPCR verifying knockdown. (n = 3, * = p values of <0.05) (B) Relative expression of DGCR8 gene in miRNAs depleted RD cells measured by qPCR verifying knockdown. (n = 3, * = p values of <0.05) (C) Relative expression of EV71 viral RNA in miRNAs depleted RKO cells measured by qPCR. (n = 3, * = p values of <0.05) (D) Relative expression of EV71 viral RNA in miRNAs depleted RKO cells measured by qPCR. (n = 3, * = p values of <0.05).
Figure 4.
Heat map of the microarray miRNA expression profile of EV71 infected and non-infected control colorectal cells, HT29.
The two-way hierarchical cluster heat map showed differential expressed miRNAs of two groups of samples. The miRNAs were chosen according to the cut off p<0.05 where blue represents miRNAs with decreased expression and red represent miRNAs with increased expression. (n = 3, * = p values of <0.05).
Figure 5.
Validation of differential expression of selected miRNAs by qPCR.
Ten expression levels of selected miRNAs from microarray assay were validated using qPCR. U6snRNA was used as reference RNA for the normalization of miRNAs and relative gene expression was quantified based on 2−ΔΔCT. (n = 3, * = p values of <0.05).
Figure 6.
Heat map of differential expression of selected miRNAs by microarray.
The two-way hierarchical cluster heat map showed differential expressed selected miRNAs of two groups of samples. The miRNAs from both methodology qPCR data showed similar direction of response in microarray analysis data with both methodologies showing similar trends where blue represents miRNAs with decreased expression and red represent miRNAs with increased expression.
Figure 7.
EV71 is unable to down regulate host antiviral response in miRNA depleted cells during infection.
DGCR8 knockdown cells and control cells (scrambled DsiRNA) were infected with EV71 (MOI of 1). Total intracellular RNA were harvested at 36 hpi, converted to cDNA and measured by quantitative real time polymerase chain reaction (qPCR) (n = 3, * = p values of <0.05). A) Relative expression of Toll-like receptor signalling measured by qPCR. (B) Relative expression of RIG-1 like receptor signalling measured by qPCR. (C) Relative expression of Nod-like receptor signalling measured by qPCR. (D) Relative expression of Type 1 interferon signalling measured by qPCR.
Figure 8.
Schematic illustration of the interplay between EV71 and host miRNAs.
EV71 altered host miRNAs such as has-miR-548 to manipulate host antiviral responses such as Toll-like signalling, NOD-like signalling, RIG-1 signalling and Type I interferon pathways to enhance its survival during pathogenesis.