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. 2018 Mar 20;9:93. doi: 10.3389/fgene.2018.00093

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Copyright © 2018 Riad, Elhelw, Shawer, El-Ekiaby, Salah, Zekri, Esmat, Amleh and Abdelaziz.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Impact of miR-155 and miR-182 on CLDN1 expression in infected Huh7 cell models. (A) A significant upregulation in the expression level of miR-155 (p = 0.0307*) and miR-182 (p = 0.0127*) is observed upon mimicking of Huh7 cells as compared to untransfected cells. A non-significant reduction in miRNA expression is observed with anti-miR-155 and a significant reduction is evident with anti-miR-182 (p = 0.0210*) (B) miR-155 (p = 0.0002***) and miR-182 mmics (p = 0.0009***) decrease CLDN1 mRNA expression in JFH1-infected cells. Also in ED43/JFH1-infected cells, miR-155 (p = 0.0179*) and miR-182 (p = 0.0395*) mimics decrease CLDN1 mRNA expression. Antagomirs against both miRNAs restored CLDN1 mRNA expression during both JFH1 and ED43/JFH1 infections to a level not significantly different than untransfected cells. Knocking down CLDN1 using siRNAs as a positive control decreases the expression of CLDN1 mRNA in both JFH1- (p = 0.0203*) and ED43/JFH1- (p = 0.0339*) infected cells. Relative Quantitation (RQ) is used for expression of the results. (C) Percentage fluorescence intensity of the immunolabeled CLDN1 protein is plotted. miR-155 (p = 0.0183*) and miR-182 mmics (p = 0.0091**) decrease CLDN1 protein levels in JFH1-infected cells. miR-155 (p = 0.0119*) and miR-182 (p < 0.0001***) mimics decrease CLDN1 protein levels in ED43/JFH1-infected cells. Knocking down CLDN1 using siRNAs decreases the expression CLDN1 protein in both JFH1 (0.0049**) and ED43/JFH1 (p < 0.0001***)-infected cells. (D) Protein levels of CLDN1 in infected Huh7 cells transfected with miR-155, miR-182 mimics or CLDN1 siRNA is assessed by western blotting, and the density of the bands is measured and plotted on a graph. Levels of CLDN1 protein (Mwt: 22.7 kDa) are normalized to levels of β-tubulin (Mwt: 55 kDa) in the same wells. Cells transfected with miR-155 (p = 0.0121*) and miR-182 mimics (p = 0.0016**) show significantly lower levels of CLDN1 protein as compared to untransfected cells. siRNAs against CLDN1 efficiently decreased its protein levels (p = 0.0022**) compared to untransfected cells. ***p < 0.001, **p < 0.01, *p < 0.05 and ns, statistically not significant.