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. 2023 Mar 8;20:62. doi: 10.1186/s12974-023-02729-6

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© The Author(s) 2023

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — THC can counteract the post-transcriptional silencing of WFS1 by miR-142-3p. The heat map shows all differentially expressed (p ≤ 0.05) miRNAs in the BG of VEH/SIV (A) and THC/SIV (B) relative to uninfected controls RMs and in VEH/SIV compared to THC/SIV RMs (C). MiRNA species originating from the opposite arm of the precursor are denoted with an asterisk (*). Red arrows (A) indicate inflammation-associated miRNAs differentially upregulated in BG of VEH/SIV RMs. Blue and green arrows indicate miRNAs commonly up and downregulated in BG of VEH/SIV (A) and THC/SIV (B) RMs, respectively. MiRNA–mRNA duplex showing a single miR-142-3p binding site on the RM WFS1 (D) mRNA 3’ UTR. RT-qPCR validation of miR-142-3p expression in BG of VEH/SIV relative to uninfected control RMs (E). Luciferase reporter vectors containing a single highly conserved miR-142-3p (F) binding site on the RM WFS1 mRNA 3′ UTR or the corresponding construct with the binding sites deleted (WFS1 DEL) were co-transfected into HEK293 cells with 30 nM miR-142-3p or negative control mimic. Firefly and Renilla luciferase activities were detected using the Dual-Glo luciferase assay system 96 h after transfection. Luciferase reporter assays were performed thrice in six replicate wells (F). Representative immunofluorescence images showing the expression of WFS1 (red) protein at 96 h post-transfection of HCN2 neuronal cells with 30 nM LNA-conjugated FAM-labeled negative control (G) or miR-142-3p (green) (H) mimics and images were quantitated (L). miR-142-3p transfected HCN2 cells were treated with DMSO (H), THC (I), AM251 + THC (J), AM630 + THC (K) 96 h post-transfection. Cells were fixed and stained after 18 h and the expression of WFS1 (red) protein and nuclear staining using DAPI (blue) were quantitated (L). Experiments were performed in triplicate wells using 3 µM of THC, 10 µM of AM251/AM630, and repeated thrice. Firefly/Renilla ratios and immunofluorescence data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test. A p-value of < 0.05 was considered significant