MiR-101-containing extracellular vesicles bind to BRD4 and enhance proliferation and migration of trophoblasts in preeclampsia

Fig. 1. HUCMSC-secreted EVs encapsulate miR-101. a Expression of CD14, CD19, CD29, CD34, CD44, CD45, CD73, CD90, and HLA-DR determined by flow cytometry. b The morphology of HUCMSCs (bar = 50 μm) observed in inverted microscope. Osteogenic, adipogenic, and cartilage differentiation was dyed with Alizarin Red S staining, Oil Red O staining, and Alcian Blue staining, respectively. c The morphology of HUCMSC-derived EV determined by TEM (100 nm). d Size distribution of the HUCMSC-exosome analyzed by NTA. e The protein levels of GM130, CD63, and TSG101 determined by immunoblotting. f The expression of miR-101 in HUCMSCs and HUCMSC-EV relative to U6 determined by RT-qPCR. *p < 0.05 vs. **p < 0.01

Fig. 2. EV-encapsulated miR-101 from HUCMSCs can be delivered into the HTR-8/SVneo cells. a Expression of miR-101 in placental tissues in patients with PE compared to healthy donors measured by RT-qPCR. b Transfection efficiency of HUCMSCs transfected with cy3-miR-101 mimic, miR-101 mimic, or miR-NC determined using RT-qPCR. c Fluorescence result of HTR-8/SVneo cells after being co-cultured with HUCMSCs transfected with miR-NC, cy3-miR-101, or cy3-miR-101 + GW4869 observed under a fluorescence microscope (scale bar = 20 μm). d Fluorescence result of HTR-8/SVneo cells after being co-cultured with EVs which were labeled with PKH67 and secreted from HUCMSCs transfected with cy3-miR-101 mimic, observed under a fluorescence microscope (scale bar = 20 μm). e Expression of miR-101 in HTR-8/SVneo cells after being co-cultured with HUCMSC-miR-NC, HUCMSC-miR-101, HUCMSC-NC-EV, and HUCMSC-miR-101-EV determined by RT-qPCR. *p < 0.05 vs. **p < 0.01

Fig. 3. EV-encapsulated miR-101 from HUCMSCs downregulates BRD4 expression in the HTR-8/SVneo cells. a Intersection of downstream regulatory genes of miR-101 predicted by TargetScan, RNAInter, and StarBase with genes related to eclampsia in the GeneCards database. b Enrichment result of candidate genes by GO analysis from the online KOBAS. The x-axis indicates the number of genes involved, while the y-axis indicates the GO entry name. The dot color indicates −log10 p value, and the dot size indicates the GO entry background gene number. c Binding relationship between miR-101 and BRD4 predicted by bioinformatic analysis. d, e mRNA and protein expression of BRD4 in the placental tissues in PE patients and healthy donors determined by RT-qPCR and immunoblotting. f Correlation between miR-101 and BRD4 in PE determined by Pearson’s correlation analysis. g The targeting relationship between miR-101 and BRD4 determined by dual luciferase reporter gene assay. h The targeting relationship between EV-encapsulated miR-101 and BRD4 determined by dual luciferase reporter gene assay. i The transfection efficiency of miR-101 in HTR-8/SVneo cells and in EVs isolated from HUCMSCs infected with miR-101…

Fig. 4. EV-encapsulated miR-101 from HUCMSCs promotes proliferation and migration of placental trophoblasts by inhibiting BRD4 expression. a Cell proliferation of HTR-8/SVneo cells in response to miR-101 mimic, miR-101 inhibitor, or their corresponding NCs determined by EDU staining (scale bar = 50 μm). b Cell migration of HTR-8/SVneo cells in response to transfection with miR-101 mimic, miR-101 inhibitor, or their corresponding NCs determined by transwell assay (scale bar = 50 μm). c Cell proliferation of HTR-8/SVneo cells in response to addition of EV released from HUCMSCs transfected with miR-101 mimic determined by EDU staining (scale bar = 50 μm). d Cell migration of HTR-8/SVneo cells in response to addition of EV released from HUCMSCs transfected with miR-101 mimic determined by transwell assay (scale bar = 50 μm). e Cell proliferation in HTR-8/SVneo cells transfected with miR-101 mimic and oe-BRD4 determined by EDU staining (scale bar = 50 μm). f Cell migration in HTR-8/SVneo cells transfected with miR-101 mimic and oe-BRD4 determined by transwell assay (scale bar = 50 μm). *p < 0.05 vs. **p < 0.01. M.NC, negative control mimic; M.miR-101, microRNA-101 mimic; I.NC, negative…

Fig. 5. BRD4 downregulation promotes proliferation and migration of placental trophoblasts by inhibiting NF-κB/CXCL11 axis. BRD4 was overexpressed or repressed in HTR-8/SVneo cells. a Overexpression and silencing efficiency of BRD4 in HTR-8/SVneo cells determined by RT-qPCR and Western blot analysis. b Cell proliferation of HTR-8/SVneo cells after treatments determined by EDU staining (scale bar = 50 μm). c Cell migration of HTR-8/SVneo cells after treatments determined by transwell assay (scale bar = 50 μm). d Venn map of intersected genes between NF-KB-related co-expression genes retrieved from MEM and eclampsia-related genes in the GeneCards database. e Network diagram of co-expression relationships among 57 candidate genes in the Coexpedia. f Expression of NF-KB, CXCL11 IL-6, TNF-α, p65, p-IkBα, and IkBα in HTR-8/SVneo cells. g The expression of NF-KB, CXCL11 IL-6, TNF-α, p65, p-IkBα, and IkBα in HTR-8/SVneo cells in response to mimic NC, miR-101 mimic, miR-101 mimic + control, and miR-101 mimic + oe-BRD4. *p < 0.05 vs. **p < 0.01. M.NC, negative control mimic; M.miR-101, microRNA-101 mimic; I.NC, negative control inhibitor; I.miR-101, miRNA-101 inhibitor; si-BRD4, short interfering…

Fig. 6. EV-encapsulated miR-101 attenuates PE by repressing BRD4 expression via inhibition of NF-κB/CXCL11 axis. a Blood pressure observed at the 15th, 17th, and 19th day in PE modeled rats, healthy rats and sham-operated rats. b 24 h urine protein observed at the 15th, 17th and 19th day in PE modeled rats, healthy rats and sham-operated rats. c Overexpression efficiency of miR-101 in HUCMSCs and HUCMSC-derived EVs determined using RT-qPCR. d Blood pressure observed at the 15th, 17th and 19th day in PE modeled rats, healthy rats, sham-operated rats, PE modeled rats treated with NC-EV, or miR-101-EV. e 24 h urine protein observed at the 15th, 17th, and 19th day in PE modeled rats, healthy rats, sham-operated rats, PE modeled rats treated with NC-EV, or miR-101-EV. f–h The weight of fetus and placenta in PE modeled rats, healthy rats, sham-operated rats, PE modeled rats treated with NC-EV, or miR-101-EV. i Placenta stained by HE staining in PE modeled rats, healthy rats, sham-operated rats, PE modeled rats treated with NC-EV, or miR-101-EV (scale bar = 25 μm). j Expression of miR-101 in PE modeled rats, healthy rats, sham-operated rats, PE modeled rats treated with NC-EV, or miR-101-EV…

Fig. 7. Schematic diagram of the proposed mechanism that EV-encapsulated miR-101 released from HUCMSCs promotes proliferation and migration of trophoblasts by downregulating BRD4 via inhibition of NF-κB/CXCL11, thereby attenuating PE