EV71 upregulates the expression of miR-16-5p through caspase-dependent pathways. (a,b) EV71-induced upregulation of miR-16-5p is inhibited by pan-caspase inhibitor, Z-VAD-FMK. CCF-STGG1 cells were treated with 20 μM Z-VAD-FMK or DMSO as control for 3 h prior to EV71 infection. After EV71 infection for 0, 3, 6, 9 and 12 h, the cells were collected for RNA extraction. The expression levels of miR-16-5p (a) and pri-miR-16-1 (b) were quantified using qRT-PCR. (c) Z-VAD-FMK treatment inhibits EV71-induced CCF-STTG1 apoptosis. CCF-STTG1 cells were pre-treated with Z-VAD-FMK and infected with EV71 as described above. After EV71 infection for 0, 6 and 9 h, cells were observed, and pictures of dishes were obtained using a phase-contrast microscope (Nikon Diaphot 300). (d,e) Analysis EV71-induced apoptosis in CCF-STTG1 cells using flow cytometry. CCF-STTG1 cells were pre-treated with 20 μM Z-VAD-FMK or DMSO as control for 3 h prior to EV71 infection and infected with EV71. After EV71 infection for 12 h, the cells were harvested and resuspended in binding buffer. The cell suspension was incubated with 5 μL Annexin V-FITC and 10 μL PI for 15 min in a dark place. Then flow cytometry (BD Biosciences, USA) was used to determine apoptosis of the CCF-STTG1 cells (d). Annexin V-FITC positive cells were expressed as mean ± SD from three different replicates (e). (f,g) Analysis the influence of EV71 non-structural proteins on the expression of miR-16-5p. 293T cells were transfected with eight non-structure proteins and empty vector PCAGGS. At 24 h post-transfection, cells were collected, and the expression levels of miR-16-5p (f) and pri-miR-16-1 (g) were examined by qRT-PCR. (h,i and j) EV71 2B, 2BC and 3C proteins can induce cell apoptosis. At 24 h post-transfection with 2B, 2BC, 3C and empty vector PCAGGS, the 293T cells were observed, and pictures of dishes were obtained by using a phase-contrast microscope (h). Then the transfected cells were harvested and resuspended for flow cytometry analysis with Annexin V/PI apoptosis assay kits (i). Annexin V-FITC positive cells were collected from three different replicates for statistical analysis (j). (k,l) 2B, 2BC, 3C-induced upregulation of miR-16-5p are inhibited by pan-caspase inhibitor, Z-VAD-FMK. 293T cells were transfected with 2B, 2BC, 3C and empty vector PCAGGS respectively. At 24 h post-transfection, cells were collected, and the expression levels of miR-16-5p (l) and pri-miR-16-1 (k) were examined by qRT-PCR. Data in (a,b,e,f,g,j,k and l) shown are the means and standard deviations (mean ± SD) and analyzed by Student’s t test. Asterisks denote significant differences between indicated samples (*p < 0.05, **p < 0.01).