CircPTPN11 inhibits the replication of Coxsackievirus B5 through regulating the IFN-I pathway by targeting miR-152-3p/SIRPA axis

Figures

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  Fig. 1. Characterization of CircPTPN11 in RD cells. (A) The levels of 10 candidate circRNAs identified from the RNA-seq analysis. (B) RT-qPCR was performed to confirm the levels of 10 candidate circRNAs according to RNA-seq results. (C) The genomic location and junction of CircPTPN11. (D) Sanger sequencing confirmation of the head-to-tail splicing of CircPTPN11. (E) RT-PCR confirmation of the head-to-tail splicing of CircPTPN11. (F) The level of CircPTPN11 with and without RNase R treatment. (G) The sub-cellular distribution of CircPTPN11 in the RD cells with or without CVB5 infection. Data are representative of three independent experiments and are plotted as the mean ± S.D. *P < 0.05, ***P < 0.001 vs. the control group.

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  Fig. 2. The levels of CircPTPN11 are induced by viral infection. (A) RD cells were infected with CVB5 (MOI=1) for the indicated times. RT-qPCR and gel-electrophoresis were performed to determine the levels of CircPTPN11. (B) RD cells were infected with CVB5 at varying MOIs for 24 h. RT-qPCR and gel-electrophoresis were performed to determine the levels of CircPTPN11. (C) RD cells were transfected with different amounts of cellular or viral RNAand harvested at 24HPI. The levels of CircPTPN11 were analyzed by qRT-PCR. (D) Various cell lines were infected with CVB5 (MOI=1) for 24 h. Cells were harvested and the levels of CircPTPN11 were analyzed by qRT-PCR. (E) Three-day-old BALB/c mice were intraperitoneally injected with CVB5, and tissue samples were collected 10 days later. RT-qPCR was performed to determine the levels of CircPTPN11 in different tissues. Data are representative of three independent experiments and are plotted as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group.

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  Fig. 3. CircPTPN11 inhibits CVB5 replication. (A-C) RD cells were transfected with either pcDNA3.1-CircPTPN11 or pcDNA3.1 (an empty vector), and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection. Cells and supernatants were harvested at 24HPI, and the levels of CVB5 VP1 were analyzed by qRT-PCR (A), and Western blotting (B). CVB5 titers were analyzed by TCID50 assay (C). (D-F) RD cells were transfected with either siCircPTPN11 or siNC (an empty vector), and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection. Cells and supernatants were harvested at 24HPI, and the levels of CVB5 VP1 were analyzed by qRT-PCR (D), and Western blotting (E). The CVB5 titers were analyzed by TCID50 assay (F). Data are representative of three independent experiments and are plotted as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group.

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  Fig. 4. CircPTPN11 activates promotes IFN-I via IRF3 to promote the expression of ISGs. RD cells were transfected with either pcDNA3.1-CircPTPN11 or pcDNA3.1 (an empty vector), and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection. Cells and supernatants were harvested at 24 HPI. (A) The secretion of IFN-α2 and IFN-β were determined by ELISA. (B) The expression of vital ISGs and IFN-I mRNA were determined by RT-qPCR. (C) The expression of MDA5, RIG-I, IKKε/p-IKKε, TBK1/p-TBK1 and IRF3/p-IRF3 were determined by Western blotting. (D) The expression of (p-IRF3)2 was determined by Western blotting. (E) The expression of IRF3 and p-IRF3 in nuclear and cytoplasmic fractions were determined by Western blotting. (F) The expression of p-IRF3 in nuclear and cytoplasmic fractions were visualized by Immunofluorescence assay. Data are representative of three independent experiments and are plotted as the mean ± S.D. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the control group.

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  Fig. 5. miR-152-3p is targeted by CircPTPN11 and promotes CVB5 replication. (A) RD cells were transfected with either pcDNA3.1-CircPTPN11 or pcDNA3.1 (an empty vector), and then the cells were harvested at 24 h post-transfection. The levels of miRNA were determined by RT-qPCR. (B) RD cells were transfected with either siCircPTPN11 or siNC (an empty vector), and then the cells were harvested at 24 h post-transfection. The levels of miRNA were determined by RT-qPCR. (C) The prediction of the binding site of miR-152-3p on CircPTPN11. The binding site sequences in CircPTPN11 were mutated, and the construct was named CircPTPN11-Mut. (D) HEK 293T cells were transfected with miR-152-3p, pmirGLO-CircPTPN11-WT or pmirGLO-CircPTPN11-Mut, and then the cells were harvested at 24 h post-transfection. The luciferase reporter assay was performed to measure luciferase activity. (E) RD cells were transfected with either miR-152-3p-inhibitor or miR-152-3p-mimic, and then the cells were harvested at 24 h post-transfection. The levels of CircPTPN11 were determined by RT-qPCR. (F-G) RD cells were transfected with miR-152-3p-inhibitor, and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection.…

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  Fig. 6. SIRPA is a downstream target gene of miR-152-3p and suppresses CVB5 replication. (A) Downstream targets genes of miR-152-3p were identified by database screening. (B) RD cells were transfected with miR-152-3p-mimic, and then the cells were harvested at 24 h post-transfection. The expression of SIRPA was determined by Western blotting. (C) RD cells were transfected with miR-152-3p- inhibitor, and then the cells were harvested at 24 h post-transfection. The expression level of SIRPA was determined by Western blotting. (D) The prediction binding site of miR-152-3p on SIRPA. Their binding site sequences in SIRPA were mutated and the construct was named CircPTPN11-Mut. (E) HEK 293T cells were transfected with miR-152-3p, SIRPA-WT or SIRPA-Mut, and then the cells were harvested at 24 h post-transfection. The luciferase reporter gene assay was performed to measure luciferase activity. (F-G) RD cells were transfected with either pcDNA3.1-SIRPA or pcDNA3.1 (an empty vector), and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection. Cells were harvested at 24 HPI. The expression of IRF3/p-IRF3 and VP1 were determined by Western blotting (F), the levels of vital ISGs, IFN-β…

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  Fig. 7. Inhibition of CVB5-Induced ISGs by the CircPTPN11/miR-152-3p/SIRPA Axis. (A) RD cells were transfected with either siCirPTPN11 or siNC (an empty vector), and then the cells were harvested at 24 h post-transfection. The expression of SIRPA were determined by Western blotting. (B) RD cells were transfected with either pcDNA3.1-CirPTPN11 or pcDNA3.1 (an empty vector), and then the cells were harvested at 24 h post-transfection. the expression of SIRPA were determined by Western blotting. (C-D) RD cells were transfected with pcDNA3.1-CircPTPN11 and/or miR-152-3p-mimic, and then the cells infected with CVB5 (MOI=1) at 24 h post-transfection. Cells were harvested at 24 HPI. The levels of OASL, ISG20, IFN-β and VP1 mRNA were determined by RT-qPCR (C), the expression of SIRPA, IRF3/p-IRF3 and VP1 were determined by Western blotting (D). Data are representative of three independent experiments with similar results. Data are representative of three independent experiments and are plotted as the mean ± S.D. *P < 0.05, **P < 0.01 vs. the control group.

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  Fig. 8. A proposed model for CircPTPN11 inhibition CVB5 replication. CircPTPN11 acts as a sponge for miR-152-3p, inhibiting the replication of CVB5 by up-regulating SIRPA via IRF3-mediated IFN-I pathway.