Hsa-let-7c-5p augments enterovirus 71 replication through viral subversion of cell signaling in rhabdomyosarcoma cells

Figures

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  Fig. 1. Relative expression of mature hsa-let-7c-5p is regulated by EV71 infection. RD cells were infected with EV71 (MOI = 5) or not (mock infection) at the indicated time points. The expression of hsa-let-7c-5p was detected by stem-loop qRT-PCR and normalized to that of U6 snRNA. The data are presented as the mean ± SD from one representative experiment, and all results here and in all subsequent figures were replicated three times. Significant differences are indicated as follows: ***p < 0.001

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  Fig. 2. EV71 replication is regulated by the hsa-let-7c-5p mimic. a The efficiency of transfection of the hsa-let-7c-5p mimic (100 nM) into RD cells for 48 h was determined by stem-loop qRT-PCR. b The EV71 titer is altered by the hsa-let-7c-5p mimic. RD cells were transfected with the NC (100 nM), hsa-let-7c-5p (100 nM) or neither (mock transfection, Mock-T) for 48 h, followed by EV71 infection (MOI = 5) for 24 h. Total virus was collected from infected cells and culture supernatants and used in plaque assays. The virus titers in the cultures are expressed as plaque-forming units (PFU) per milliliter. c The expression of viral RNA and (d) the viral structural protein VP1 was assessed by qRT-PCR and Western blotting, respectively, after transfection of cells with the hsa-let-7c-5p mimic, and GAPDH was used as an internal control. RD cells were transfected with the NC (100 nM), hsa-let-7c-5p (100 nM) or neither for 48 h, followed by EV71 infection (MOI = 5) for 24 h. In addition, RD cells were subjected to mock infection, as shown in (d), to assess specificity of the VP1 primary antibody. The lower panel in (d) shows that VP1 expression normalized to GAPDH is expressed as a fold change…

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  Fig. 3. EV71 replication is modulated by the hsa-let-7c-5p inhibitor. a The effect of transfection of hsa-let-7c-5pi (150 nM) into RD cells for 48 h on hsa-let-7c-5p expression was assessed by stem-loop qRT-PCR. b The virus titer is regulated by hsa-let-7c-5pi. RD cells were transfected with NCi (150 nM), hsa-let-7c-5pi (150 nM) or neither for 48 h and then infected with EV71 (MOI = 5) for 24 h. Total virus was collected from infected cells and culture supernatants and used in plaque assays. The virus titers in the cultures are expressed as PFU per milliliter. c Effects of the hsa-let-7c-5p mimic on the expression of viral RNA and (d) the viral structural protein VP1 were analyzed by qRT-PCR and Western blotting, respectively, using GAPDH as an internal control. RD cells were transfected with NCi (150 nM), hsa-let-7c-5pi (150 nM) or neither for 48 h, followed by EV71 infection (MOI = 5) for 24 h. The RD cells were subjected to mock infection, as shown in (d), to assess the specificity of the VP1 primary antibody. The lower panel in (d) shows that VP1 expression normalized to GAPDH is expressed as a fold change compared with the Mock-T group, as quantified using Image J software. e The…

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  Fig. 4. Target of hsa-let-7c-5p in MAP4K4. a Predicted binding site of hsa-let-7c-5p to the 3′UTR of human MAP4K4, as determined using TargetScan. b MAP4K4 was validated as a target of hsa-let-7c-5p by dual luciferase reporter assay. RD cells were transfected with a wild-type or mutant 3′UTR vector (50 ng) for 6 h, followed by transfection of the NC (100 nM) or hsa-let-7c-5p mimic (100 nM) for 48 h. Renilla luciferase activity in each sample was normalized to Firefly luciferase activity. c The effect of the hsa-let-7c-5p mimic on the MAP4K4 mRNA level. RD cells were transfected with NC (100 nM) or hsa-let-7c-5p (100 nM) for 48 h. RNA was extracted and analyzed by qRT-PCR. d Regulation of the MAP4K4 protein level by the transfection of RD cells with the hsa-let-7c-5p mimic (100 nM) for 48 h, as demonstrated by Western blotting. The right panel in (d) shows that MAP4K4 expression normalized to GAPDH is expressed as a fold change compared with the NC mimic group, as quantified using Image J software. e The MAP4K4 mRNA level is affected by hsa-let-7c-5pi. RD cells were separately transfected with NCi (150 nM) and hsa-let-7c-5pi (150 nM) for 48 h, and then RNA was analyzed by qRT-PCR. f…

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  Fig. 5. The effect of MAP4K4 knockdown on EV71 replication. a qRT-PCR and, b Western blotting were performed to assess the expression of MAP4K4 in response to EV71 infection in RD cells. RD cells were infected with or without EV71 at 5 MOI at the indicated time points. The right panel in (b) shows that the MAP4K4 protein level normalized to GAPDH is expressed as a fold change compared with the mock infection (24 h) group, as quantified using Image J software. c The effect of MAP4K4 knockdown on the virus titer was determined at 24 h p.i. by plaque assays. RD cells were transfected with the LV-shcontrol or LV-shMAP4K4-3 vector for 48 h, followed by infection with EV71 (MOI = 5) for 24 h. Total virus was collected from infected cells and culture supernatants and used in plaque assays. The virus titers in the cultures are expressed as PFU per milliliter. d Viral RNA and MAP4K4 mRNA expression and (e) VP1 protein and MAP4K4 protein expression following MAP4K4 knockdown were determined by qRT-PCR and Western blotting, respectively. The LV-shcontrol or LV-shMAP4K4-3 vector was transfected into RD cells for 48 h, followed by EV71 infection (MOI = 5) for 24 h. The right panel in (e) shows that…

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  Fig. 6. Regulation of the JNK signaling pathway by MAP4K4 and hsa-let-7c-5p. a The LV-shMAP4K4-3 vector, b hsa-let-7c-5p mimic (100 nM), c hsa-let-7c-5pi (150 nM) or corresponding control was transfected into RD cells for 48 h. The total JNK1/2 and p-JNK1/2 protein levels were analyzed by Western blotting. The ratios of the p-JNK1/2 to total JNK1/2 signals are expressed as fold changes compared with the corresponding controls, as quantified using Image J software (lower panels). *p < 0.05; **p < 0.01

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  Fig. 7. Modulation of JNK pathway activity by EV71 infection. a The effect of EV71 infection on JNK activity was assessed by Western blotting. RD cells were infected with EV71 (MOI = 5) at the indicated time points, and the data were analyzed by comparison with the mock-infected group. The ratios of the p-JNK1/2 to total JNK1/2 signals are expressed as fold changes compared with the mock-infected group, as quantified using Image J software, as shown in (b). c The effect of UV inactivation of EV71 on JNK activity was assessed by Western blotting. RD cells were infected with UV-inactivated EV71 at the indicated time points, and the data were analyzed by comparison with the mock-infected group. The ratios of the p-JNK1/2 to total JNK1/2 signals are expressed as fold changes compared with the mock-infected group, as quantified using Image J software, as shown in (d). *p < 0.05; **p < 0.01

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  Fig. 8. Impact of the JNK inhibitor SP600125 on EV71 replication. a Effect of SP600125 on the CPE of EV71-infected RD cells. RD cells were pretreated with or without SP600125 for 1 h and infected with EV71 (MOI = 5) for 12, 24 or 36 h. Cell morphology was examined by light microscopy (40×). The CPE is labeled with red arrows. The scale bar is 100 μm, as shown in the first image. b Regulation of the virus titer by SP600125 at 24 h p.i. was assessed by plaque assays. The virus titers in the cultures are expressed as PFU per milliliter. c Regulation of the expression of EV71 RNA by SP600125 at 24 h p.i. was examined by qRT-PCR. d The effects of SP600125 on the VP1, total JNK1/2 and p-JNK1/2 protein levels were assessed by Western blotting. RD cells were pretreated with or without SP600125 for 1 h prior to EV71 infection (MOI = 5) or mock infection for 24 h. **p < 0.01; ***p < 0.001