For instance, miR-101 represses feline herpesvirus 1 reproduction by targeting cellular suppressor of cytokine signaling 5 to promote IFN-I signaling [20]; miR-7 suppresses rotavirus replication via targeting viral NSP5 directly [21]; miR-122 reduces HBV gene expression and replication via binding with hepatitis B computer virus pre-genomic RNA sequence [22]; miR-21-3p promotes influenza A computer virus H5N1 replication by regulating FGF2 to repress IFN-I signaling [23]; miR-545 promotes enterovirus 71 propagation through directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6 [24]
For instance, miR-101 represses feline herpesvirus 1 reproduction by targeting cellular suppressor of cytokine signaling 5 to promote IFN-I signaling [20]; miR-7 suppresses rotavirus replication via targeting viral NSP5 directly [21]; miR-122 reduces HBV gene expression and replication via binding with hepatitis B computer virus pre-genomic RNA sequence [22]; miR-21-3p promotes influenza A computer virus H5N1 replication by regulating FGF2 to repress IFN-I signaling [23]; miR-545 promotes enterovirus 71 propagation through directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6 [24]. cells were challenged by FMDV (O/BY/CHA/2010) at 0.1 MOI or 1 MOI, separately. qRT-PCR was applied to quantify miR-4334-5p expression. The expression level of miR-4334-5p was rapidly upregulated at Kaempferol around 0. 5 h and continued Kaempferol increasing till 2 h, and following decreased post 4~6 h. Compared with control (0 h), the expression level of miR-4334-5p was more than 21-fold higher in cells infected with 0.1 MOI (Figure 1A) at 0.5 h post infection, and also in 1 MOI infection group it is 6-fold higher compared with control (Determine 1B). After that, the expression level of miR-4334-5p decreased constantly, at 6 h post contamination, compared with control (0 h), miR-4334-5p expression was less than 10% in cells infected with 0.1 MOI (Figure 1A), and the level decreased to 15% in 1 MOI infection group (Figure 1B). Mmp9 This sharply and dramatic change of miR-4334-5p during FMDV contamination implied it might involve in the regulation on FMDV replication. Open in a separate window Physique 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 impartial experiments with comparable results, and normalized to miR-16; Error bars is standard deviation (SD). Significance was calculated by Students t-test, *** 0.001; ** 0.01; * 0.05. 3.2. FMDV Replication Was Up-Regulated by miR-4334-5p Mimics In order to evaluate the possible regulatory function of miR-4334-5p on FMDV reproduction, the mimics and scrambled negative-control (NC) RNAs of miR-4334-5p were synthesized and transfected into PK-15 cells. As shown in Physique 2A, compared with NC groups, the miR-4334-5p expression increased more than 1000-fold at 18 h post transfection of miR-4334-5p mimics, and it increased more than 500-fold at 24 h, which clearly exhibited that this miR-4334-5p mimics work well in PK-15 cells. To explore the role of miR-4334-5p during FMDV contamination, the mimics or scrambled negative-control (NC) of miR-4334-5p were transfected into PK-15 cells separately, and then challenged with FMDV at 0.1 MOI post transfection. Compared with in the control cells, transfection of miR-4334-5p mimics promoted FMDV propagation, in Physique 2BCD, the computer virus structural protein VP1 level (qRT-PCR or Western-blot) and computer virus titers all increased significantly. All of the above data clearly revealed that this up-regulation of miR-4334-5p promotes FMDV replication. Open in a separate window Physique 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, ** 0.01. (B). MiR-4334-5p mimics or scramble Kaempferol 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 -actin. The data are means SD from triplicate assays, *** 0.001; ** 0.01 (C). Kaempferol PK-15 cells were treated as in (B), the cells were lysed and then subjected to Western blot, Kaempferol VP1 and -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 computer virus titers by TCID50 (Median Tissue Culture Infectious Dose) assay. Results shown are means SD from triplicate assays, * 0.05. 3.3. FMDV Replication Was down-Regulated by miR-4334-5p Inhibitors In order to further evaluate the possible regulatory function of miR-4334-5p on FMDV reproduction, miR-4334-5p inhibitors and scrambled negative-control (NC) RNAs were synthesized. Compared with the transfection of NC, inhibitors repressed the expression level of miR-4334-5p to less than 40%, which exhibited the knock-down efficiency is usually significant (Physique 3A). To further validate the role of miR-4334-5p on FMDV reproduction, the inhibitors and scrambled.