The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway

Figures

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  Fig. 1. CVB3 infection activates P38 MAPK pathway and upregulates miRNA-21 expression, which targets the 3′UTR of MAP2K3. HELA cells were infected with CVB3 at MOI = 10 and profiling of cellular miRNA-21 and P38 MAPK proteins expression were detected, function of miRNA-21 in targeting MAP2K3 were also validated. a miRNA-21 expression was detected by Real-time PCR in CVB3-infected HeLa cell at indicated time post infection, MOI = 10. All data were normalized to U6 RNA. b Time courses for CVB3 stimulation and phosphorylation of MAP2K3 and proteins in the P38 pathways in HeLa cells. GAPDH is shown as a loading control. Shown were representative results of 3 independent tests. c Sequence alignment of miRNA-21 with putative binding sites within the wild-type and mutant 3′-untranslated regions of MAP2K3. d Relative luciferase activity of HeLa cells transfected with wild-type or mutant MAP2K3 3′UTR luciferase reporter with miRNA-21 or a mutant miRNA-21. Results represent the means and standard deviations from three independent triplicated experiments (N = 6), (*p < 0.05)

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  Fig. 2. Inactivation of P38 MAPK and reduction of MAP2K3 could reduce CVB3 replication. Hela cells were pretreated with P38 inhibitor SB203580 or MAP2K3 specific siRNA (MAP2K3-siRNA as shown in the figure) and were then infected with CVB3 at MOI = 1. DMSO and siRNA has no targets (siRNA-con as shown in the figure) were used as control. a Western bolt was conducted to detect P-P38 and downstream P-HSP27 in the presence of SB203580 7 h post CVB3 infection, DMSO was loaded as control group. b CVB3 replication in the supernatants of SB203580 treated cells at indicated time point were detected. c Western bolt was conducted to detect MAP2K3, P-P38 and downstream P-HSP27 expression in MAP2K3 specific siRNA treated cells 7 h post CVB3 infection. siRNA has non targets to the human genome was tested as a control. Shown were representative results from 3 independent tests. d CVB3 replication in the supernatants of siRNA-MAP2K3 treated cells were detected at indicated time point. Virus titer values represent the means and standard deviations of three independent experiments, (*p < 0.05)

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  Fig. 3. Exogenous miRNA-21 inhibits CVB3 replication by targeting MAP2K3 in HeLa cells. HeLa cell were transduced with Lenti-miRNA-21 at MOI = 10, and were then infected with CVB3 at MOI = 1 3 days later (miR21-up as shown in the figure). Cells transduced with Lenti-CON followed by CVB3 infection were used as control group (miR-con as shown in the figure). a miRNA-21 expression in HeLa cells after transduction with Lenti-miRNA-21 or control lentivirus 3 days and 5 days later, miR-con represents cells transduced with Lenti-CON and miR21-up represents cells transduced with Lenti-miRNA-21. b Western blotting was conducted to detect MAP2K3, P-P38 MAPK and P-HSP27 expression in HeLa cells 7 h post CVB3 infection in Lenti-miRNA-21 transduced cells. GAPDH was loaded as a control group. Shown were representative results from 3 independent test. c CVB3 replication in the supernatants post CVB3 infection at indicated time point in Lenti-miRNA-21 transduced cells. Virus titer values represent the means and standard deviations of three independent experiments, (*p < 0.05)

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  Fig. 4. The effect of miRNA-21 on viral replication and host cell apoptosis and proliferation. HeLa cells were transduced with Lenti-miRNA-21 at MOI = 10, followed by CVB3 infection at MOI = 1 at 3 days after transduction. Cells transduced with Lenti-miR-CON and sham-infected cells were used as controls. a Western blot assay was conducted to detect CVB3 capsid protein VP1 at 12 h post infection. Representative results from 3 independent experiments are shown. b Intracellular viral titers were detected at 12 h post CVB3 infection. c Cell lysates were harvested and caspase-3 activity was detected using a fluorogenic substrate at indicated time points. d Immunoblot analysis of whole-cell protein lysates from CVB3 infected cells probed with cleave-caspase 3 and Bax antibody. e CVB3 induces apoptosis was measured by Annexin-V binding to externalized phosphatidylserine, representative FACS data of annexin-V+ apoptotic cells were shown. Cell viability assay was performed at multiple time points, with different amounts of miRNA-21 without (f) or with CVB3 infection (g). The viability of HeLa cell was determined by MTS assay at indicated time points, (*p < 0.05)

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  Fig. 5. miRNA-21 overexpression inhibits CVB3 pathogenesis in mice. To detect whether high levels of miRNA-21 have effect on CVB3 infected mice, 2 × 108 transduction units of lenti-miRNA-21 were intravenously injected into mice via the caudal vein. Each mice were then inoculated intraperitoneally with a dose of 1 × 105 plaque forming units (PFU, LD50) of CVB3 virus (miR21-up as shown in the figure). Mice transduced with Lenti-CON followed by CVB3 infection were used as control group (miR-con as shown in the figure). A Lenti-miRNA-21 were intravenously injected into mice and miRNA-21 levels were increased significantly in the heart. B CVB3 viral titers detection were conducted at indicated time point in the hearts of mice after treatment with Lenti-miRNA-21 (n = 5), and viral titers were decreased compared with control group. C Western blot of MAP2K3, P-P38 MAPK, and P-HSP 27 expression in the heart of mice transduced with Lenti-miRNA-21. GAPDH was loaded as control group. Shown were representative results from 3 independent tests. D TUNEL assay were performed at day 7 post infection and percentage of TUNEL positive cells in each sample were shown, representative images were shown,…