microRNA-4516 Contributes to Different Functions of Epithelial Permeability Barrier by Targeting Poliovirus Receptor Related Protein 1 in Enterovirus 71 and Coxsackievirus A16 Infections

Figures

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  Figure 1. Viral growth curves and infection rates of EV-A71 and CV-A16 in 16HBE cells at an MOI of 10. (A) The replication kinetics of EV-A71 and CV-A16 were determined by qRT-PCR. (B) Infectious virus particles from EV-A71- and CV-A16-infected cells were quantitated by a traditional plaque assay. (C) Expression of EV-A71/CV-A16-VP1 protein in 16HBE cells subjected to EV-A71 and CV-A16. (D) The statistical results of EV-A71/CV-A16-VP1 protein expression displayed as a histogram. Significant differences among these groups are indicated by *P < 0.05. (E) The efficacy of EV-A71 and CV-A16 infections was measured by an immunofluorescence assay over a period of 12 h. (F) The rate of EV-A71 and CV-A16 infection was evaluated quantitatively by flow cytometry. (G) The data of flow cytometry analysis represented as a line chart. In (A,B,D,G) the results are the averages of triplicate samples. Error bars represent the standard errors of the mean (SEM).

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  Figure 2. Increased epithelial permeability due to EV-A71 and CV-A16 infections in 16HBE cells. (A) Morphological changes of 16HBE cells affected by EV-A71 and CV-A16 infections at 6 and 12 hpi. The yellow arrow indicates the enlarged intercellular gaps. (B) Epithelial permeability was detected by a paracellular FITC-dextran flux assay. Significant differences among these groups are indicated by *P < 0.05. (C) EV-A71 and CV-A16 infections promote 16HBE cell apoptosis, including early apoptotic and late apoptotic.

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  Figure 3. Measurement by qRT-PCR of adhesion-related molecules in 16HBE cells following EV-A71 and CV-A16 infections. The expression levels of adhesion-related genes mRNA were normalized to the constitutive expression level of GAPDH mRNA. Data in all panels are representative of at least 3 separate experiments.

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  Figure 4. PVRL1 was speculated as the direct target of miR-4516. (A) The only miRNAs identified by the three adhesion-related GOs were considered as key miRNAs for in-depth research. The miRNAs highlighted in red and blue represent the up-regulation and down-regulation in the EV-A71-infected groups, respectively, indicating adverse regulation in the CV-A16-infected groups, respectively. The miR-4516 was chosen for further study due to its most significant change. (B) miR-4516 and PVRL1 expression were detected by qRT-PCR array in 16HBE cells with EV-A71 and CV-A16 infection. (C) The target interaction between miR-4516 and PVRL1 was determined by a dual-luciferase reporter assay. Significant differences among these groups are indicated by **P < 0.01 and *P < 0.5. (D) WB analysis of PVRL1 expression in transfected cells. Transfection with the miR-4516-overexpression plasmid resulted in a significant reduction of PVRL1 protein expression in 16HBE cells. GAPDH was used as a reference.

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  Figure 5. Confocal imaging showed the junctional protein localization of nectin1 (A), claudin4 (B), E-cadherin (C), and ZO-1 (D) in differently treated 16HBE cells. The si-control and miR-4516-knockdown plasmids contain a GFP tag (green color). EV-A71/CV-A16-VP1, junctional proteins and the cell nucleus are indicated by red, purple and mazarine, respectively. The white arrows indicate that the transfected cells with si-control still show a normal “chicken wire” appearance of the above junctional proteins. The yellow arrows indicate that the EV-A71-infected cells displayed slighter changes of these proteins. The light blue arrows represent that the EV-A71-infected cells with miR-4516-overexpression plasmid pretreatment showed remarkably lower levels of these proteins. The rose red arrows denote that the CV-A16-infected cells also show a large reduction of these proteins. The saffron arrows indicate that CV-A16-infected cells pre-treated with the miR-4516-knockdown plasmid show less recovery of these proteins.

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  Figure 6. Junctional proteins expression in 16HBE cells infected with EV-A71 or EV-A71 pre-treated with a miR-4516-overexpression plasmid, and CV-A16, or CV-A16 pre-treated with a miR-4516-knockdown plasmid. (A) Protein expression of junctional proteins in 16HBE cells with different treatments normalized against GAPDH. (B) Densitometry of WBs of three independent experiments performed with lysates at 12 hpi. The signal intensity was normalized to GAPDH levels from the same blots. The results are expressed as the mean± SEM for three subjects. The statistical significance in panel B was assessed with one-way ANOVA followed by a Bonferroni post hoc test. Asterisks indicate statistical significance (*P < 0.05).