Fig. 4.

HucMSC-exos upregulated the miR-146a-5p expression which targets the TRAF6 and NF-κB signaling pathway in vitro. A Relative expression level of miRNAs analyzed by qRT‒PCR in the HTR8/SVneo cells in the aPL group and aPL + EXO group (the red box represent the HTR8/SVneo cells treated with aPL + EXO, the blue box represent the HTR8/SVneo cells treated with aPL) (n = 3). B Relative mRNA expression level of TRAF6 in the HTR8/SVneo cells analyzed by qRT‒PCR (n = 6). C StarBase analysis of the target genes of miR-146a-5p. D Schematic diagram depicted the predicted binding site of miR-146a-5p targeting the 3'-UTR of TRAF6. E Luciferase reporter gene assay of miR-146a-5p mimic treated HEK293T cells, which overexpressed either TRAF6-wildtype 3'UTR or TRAF6 -mutant 3'UTR (n = 3). F-L HTR8/SVneo cells were transfected with miR-146a-5p mimic or inhibitor before the aPL-injury process. F The relative mRNA expression level of miR-146a-5p in HTR8/SVneo cells transfected with miR146a-5p mimic (NC mimic) or inhibitor (NC inhibitor) were detected by qRT-PCR (n = 3). G The relative mRNA expression level of TRAF6 in HTR8/SVneo cells transfected with miR146a-5p mimic (NC mimic) or inhibitor (NC inhibitor) was detected by qRT-qPCR (n = 6). H, I and K Western blot analyzed the relative protein levels of TRAF6 and NF-κB p65 in HTR8/SVneo cells transfected with miR146a-5p mimic (NC mimic) or inhibitor (NC inhibitor), quantified by signal intensity normalized to ACTIN (n = 5). J and L Immunofluorescence (IF) analyzed the nuclear translocation of NF-κB p65 in the HTR8/SVneo cells transfected with miR-146a-5p mimic (NC mimic) or miR-146a-5p inhibitor (NC inhibitor), (NF-κB p65, green; Actin, red; DAPI nulcear stain, blue; Scale bars, 20 µm), (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001