Figure 6.

miR-26a-5p derived from MSC-EVs inhibited pro-inflammatory/fibrotic gene expression in vitro

(A) Heatmap showing the expression of pro-fibrotic and inflammatory markers in sham, IRI-1 d, IRI-3 d, and IRI-7 d kidneys, determined by bulk sequencing. (B and C) Characterization of MSC-EVs by electron microscopy (B) and western blotting (C). (D) Expression of miRNA-26a-5p in HK2 cells, MSCs, and MSC-EVs, determined by PCR. (E) Internalization of Dil-labeled EVs (red) by HK2 cells. (F) Western blotting and semiquantitative analysis showing the levels of ZEB2 protein in H/R-HK2 cells +/− administration of MSC-EVs. (G) Detection of internalized Cy3-miR-26a-5p (red) in MSC-EV-treated HK2 cells. (H) Results of luciferase reporter assays following co-transfection of miR-26a-5p or negative control (NC) with a Zeb2 3′ UTR reporter (H27161) or control reporter (H306) in 293T cells. (I) Transfection efficiency of miR-26a-5p mimic in HK2 cells was determined by PCR. (J) Detection of transfected Cy3-labeled miR-26a-5p mimic (red) in HK2 cells. (K) Western blotting showing ZEB2 and pi-NF-κB protein levels in HK2 cells undergoing H/R +/− exogenous miR-26a-5p. Data are expressed as mean ± SD (n = 3); Student’s t test was used for comparisons of two groups; one-way ANOVA was used for comparisons of three or more groups. ∗p < 0.05, ∗∗p < 0.01.