miR-196a-5p-Rich Extracellular Vesicles from Trophoblasts Induce M1 Polarization of Macrophages in Recurrent Miscarriage

Figure 5

Trophoblast-derived EV miR-196a-5p promotes M1 polarization of macrophages via an IκBα-mediated NF-κB pathway. (a) Predicted miR-196a-5p binding sequence on the 3-UTR of IκBα. (b) The effects of overexpression or knockdown of miR-196a-5p on the luciferase activity of IκBα reporter gene in 293T cells. (c) Luciferase activity of IκBα reporter in 293T cells treated with EVs (25 μg/ml) derived from miR-196a-5p overexpressing or knockdown HTR-8 cells. (d) PMA-pretreated THP-1 cells were transfected with biotin-labeled miR-NC or miR-196a-5p for 48 h. Interaction of IκBα transcripts with miR-196a-5p was analyzed using a pull-down assay. (e) qPCR analysis of IκBα expression levels in the PMA-pretreated THP-1 cells after transfecting with miR-196a-5p mimics or anti-miR-196a-5p. (f) The changes in IκBα mRNA expression in PMA-pretreated THP-1 cells treated with EVs (25 μg/ml) derived from miR-196a-5p overexpressing or knockdown HTR-8 cells or their respective controls. (g) Western blotting analysis of the expression levels of IκBα and nuclear p65 in PMA-pretreated THP-1 cells after transfection with miR-196a-5p mimics or anti-miR-196a-5p. (h) Western blot analysis of the expression levels of IκBα and nuclear p65 in PMA-pretreated THP-1 cells treated with EVs (25 μg/ml) derived from miR-196a-5p overexpressing or knockdown HTR-8 cells. (i–k) PMA-treated THP-1 cells were treated with miR-196a-5p mimics or EVs derived from miR-196a-5p-overexpressing HTR-8 cells and further transfected with an IκBα overexpression plasmid. (i, j) The expression levels of typical M1 markers were measured using qPCR and flow cytometry. (k) The protein expression levels of IκBα and nuclear p65 were measured using western blotting. (l) PMA-treated THP-1 cells were treated with EVs derived from miR-196a-5p-overexpressing HTR-8 cells and further treated with BAY11-7085, an inhibitor of the NF-κB pathway. The expression of typical M1 markers was measured using qPCR and flow cytometry (; ; ; ns: not significant). EV: extracellular vesicle; PMA: phorbol 12-myristate 13-acetate.

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