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

Abstract

Background: The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear.

Methods: We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus.

Results: We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time.

Conclusions: miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

Keywords: CVB3; P38 MAPK; Viral release; miRNA-21.

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)

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)

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)

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)

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, original magnification, ×200. E Immunohistochemistry for cleaved-caspase 3 at day 7 post infection were detected, representative immunohistochemistry observation of each group was shown, examples of cleaved-caspase 3 expression are shown (black arrow), original magnification, ×200. F Histological analysis in the heart tissues at day 7 post infection of mice pretreated with Lenti-miRNA-21 or control. Examples of necrosis, calcification are shown (black arrow). Original magnification, ×200. G Evaluation of the antiviral effects of Lenti-miRNA-21 on mice survival rate. Mice were pretreated with 2 × 10⁸ transduction units of lentivirus (miR21-up and miR-con as shown in the figure) or MEM (CVB3 as shown in the figure) as indicated followed by infection of LD50 CVB3 and survival was evaluated, (*p < 0.05)