MicroRNA-324-3p Plays A Protective Role Against Coxsackievirus B3-Induced Viral Myocarditis

Abstract

Viral myocarditis (VM) is an inflammatory disease of the myocardium associated with heart failure, which is caused by common viral infections. A majority of the infections are initiated by coxsackievirus B3 (CVB3). MicroRNAs (miRNAs) have a major role in various biological processes, including gene expression, cell growth, proliferation, and apoptosis, as well as viral infection and antiviral immune responses. Although, miRNAs have been found to regulate viral infections, their role in CVB3 infection remains poorly understood. In the previous study, miRNA microarray results showed that miR-324-3p expression levels were significantly increased when cells and mice were infected with CVB3. It was also found that miR-324-3p downregulated TRIM27 and decreased CVB3 replication in vitro and in vivo. In vitro, analysis of downstream signaling of TRIM27 revealed that, miR-324-3p inhibited CVB3 infection, and reduced cytopathic effect and viral plaque formation by reducing the expression of TRIM27. In vivo, miR-324-3p decreased the expression of TRIM27, reduced cardiac viral replication and load, thereby strongly attenuating cardiac injury and inflammation. Taken together, this study suggests that miR-324-3p targets TRIM27 to inhibit CVB3 replication and viral load, thereby reducing the cardiac injury associated with VM.

Keywords: Coxsackievirus B3 (CVB3); TRIM27; Viral myocarditis (VM); miR-324-3p.

Fig. 1

miR-324-3p suppresses CVB3 replication. A HeLa cells were infected with CVB3 (MOI = 10); RT-qPCR analysis was used to detect the expression level of miR-324-3p (n = 3). B HeLa cells were transfected with miRNA and cultured for 48 h, then infected with CVB3 (MOI = 10); total RNA and protein were extracted for the detection of VP1 by RT-qPCR analysis (n = 3). C, D HeLa cells were transfected with miRNA and cultured for 48 h, then infected with CVB3 (MOI = 10); total protein was extracted for the detection of VP1 by Western blot analysis. E Cytopathic effect (CPE) was observed. HeLa cells were transfected as described above and cell morphology was analyzed by microscopy after 5 h and 8 h of CVB3 infection (original magnification × 400). F Viral plaque assay. HeLa cells were transfected with miR-324-3p and miR-NC for 48 h, infected with CVB3 (MOI = 0.01) for 72 h (n = 3). G HEK-293 T cells were co-transfected with miRNA and EGFP-CVB3. A fluorescence microscope was used to observe EGFP-positive cell number after 48 h (original magnification × 100). CVB3, coxsackievirus B3; MOI, multiplicity of infection.

Fig. 2

miR-324-3p influences the expression level of VP1. A IF microscopy analysis showed the expression of VP1 in HeLa cells after overexpressing miR-324-3p and infected with CVB3 (MOI = 10) for mock, 5 h and 8 h. B IF microscopy analysis showed the expression of VP1 in HeLa cells after transfected with miR-324-3p-inhibitor and infected with CVB3 (MOI = 10) for mock, 5 h and 8 h. IF, immunofluorescence; CVB3, coxsackievirus B3; MOI, multiplicity of infection; scale bar: 10 μm.

Fig. 3

CVB3 infection and miR-324-3p regulate the expression level of TRIM27. A Prediction of miR-324-3p targeting sequence on trim27. B Validation of miR-324-3p targeting on trim27 by luciferase assay (n = 3). C, D HeLa cells were transfected with miRNA for 48 h; the expression level of TRIM27 was analyzed by Western blot and RT-qPCR (n = 3). E RT-qPCR analysis was done to measure the expression level of TRIM27 after HeLa cells infected with CVB3 (n = 3). F The protein level of TRIM27 was analyzed by Western blot after HeLa cells infected with CVB3 for 5 h and 8 h (MOI = 10). G The expression of TRIM27 in HeLa cells was analyzed by IF after infected with CVB3 for 5 h and 8 h (MOI = 10). CVB3, coxsackievirus B3; MOI, multiplicity of infection; IF, immunofluorescence; scale bar: 10 μm.

Fig. 4

TRIM27 regulates CVB3 replication through ERK1/2 signaling pathway. A HeLa cells were treated with PD98056 or DMSO for 12 h, and infected with CVB3 (MOI = 10) for 8 h. Western blot analyzed the expression levels of VP1, ERK1/2, p-ERK1/2, and TRIM27, using GAPDH as control. B, C HeLa cells were transfected with empty plasmid vector or TRIM27 vector for 24 h, treated with PD98056 or DMSO for 12 h, and infected with CVB3 (MOI = 10) for 8 h. Western blot was applied to determine the expression level of VP1, ERK1/2, p-ERK1/2, and TRIM27, using GAPDH as control. D, E HeLa cells were co-transfected with miRNA mimics and empty plasmid vector or TRIM27 vector, and infected with CVB3. The expression levels of TRIM27, VP1, ERK1/2, and p-ERK1/2 were measured by Western blot analysis, using GAPDH as a loading control. CVB3, coxsackievirus B3; MOI, multiplicity of infection.

Fig. 5

The role of TRIM27 in CVB3 replication. A, B HeLa cells were transfected with TRIM27 vector or si-TRIM27, and infected with CVB3 (MOI = 10) for 5 h and 8 h; total protein was extracted to analyze the expression level of VP1, TRIM27, ERK1/2 and p-ERK1/2, using GAPDH as a loading control. C Viral plaque assay. HeLa cells were transfected with miR-324-3p and miR-NC for 48 h, and infected with CVB3 (MOI = 0.01) for 72 h (n = 3). D HeLa cells were transfected with miRNAs and infected with CVB3 (MOI = 10) for 8 h. IF microscopy analyzed the expression of TRIM27. CVB3, coxsackievirus B3; MOI, multiplicity of infection, scale bar: 10 μm.

Fig. 6

The function of miR-324-3p on CVB3-induced VM. A RT-qPCR was employed to test the expression level of miR-324-3p in mouse heart tissues (n = 3). B The body weight of mice in different groups were recorded (n = 3). C Mouse serum was extracted to detect the level of lactate dehydrogenase (LDH) (n = 3). D, F The expression level of VP1-coding RNA was measured by RT-qPCR (n = 3). E, G The protein level of VP1 was detected by Western blot analysis (n = 3). VM, viral myocarditis.

Fig. 7

The role of miR-324-3p in VM. miR-324-3p was overexpressed or silenced by AAV injection in mice for 4 weeks. The mice were then infected with CVB3 (10⁵ PFU/mouse) for next 3 weeks. Samples were collected for analysis. A RT-qPCR analysis was used to determine the expression of TRIM27 (n = 3). B Western blot analysis was used to determine the protein levels of VP1, TRIM27 and p-ERK1/2. GAPDH was used as a loading control. C The expression level of TRIM27 in mouse heart tissue was measured by immunohistochemical (IHC) analysis (original magnification × 400). D Heart tissue sections were prepared 3 weeks post-infection (n = 3). Histopathological evaluation was performed to detect the presence and severity of myocarditis in the heart tissues of different groups (original magnification × 400). The partial enlarged image enlarges 4.125 times of the original image, and the arrow points out where the infiltrating white blood cells. VM, viral myocarditis; AAV, adeno-associated viruses; CVB3, coxsackievirus B3.