Research Paper Volume 12, Issue 4 pp 3880—3898

Adipose-derived mesenchymal stem cells-derived exosome-mediated microRNA-342-5p protects endothelial cells against atherosclerosis

Figure 3. The screened miR-342-5p facilitated the apoptosis of HUVECs impaired by H2O2. (A) The heat map of top-30 upregulated miRNAs ranked by log2FC value in RNA-sequencing analysis was generated by the online tool ClustVis. (B) The expression levels of ten candidate miRNAs were determined in HUVECs impaired by H2O2 (1500 uM) by qRT-PCR assay. The relative expression of has-miR-342-5p achieved the highest among these ten miRNAs. (C) The expression of miR-342-5p was detected with the gradually increasing concentrations of H2O2 (1000, 1500 or 2000 uM) to treat HUVECs. Results indicated that miR-342-5p expression increased along with the increasing concentration of H2O2. (D) HUVECs were separately transfected with miR-342-5p mimic, inhibitor and their corresponding NC, followed by treating with 1500 uM of H2O2. Their apoptotic rates were then analyzed by flow cytometry assay. The apoptosis rate was increased in miR-342-5p mimic group (miR-342-5p mimic+H2O2) and decreased in miR-342-5p inhibitor group (miR-342-5p inhibitor+H2O2) when separately compared to their corresponding NC group. (E) HUVECs were separately transfected with miR-342-5p mimic, inhibitor and their corresponding NC, followed by treating with 1500 uM of H2O2. The well-known apoptin (PARP, caspase 3, cytochrome C, Bcl-2, Bax and p53) in treated HUVECs was evaluated by western blot assay. The expression levels of cleaved-PARP/PARP, cleaved-caspase3/caspase3, cytochrome C, and p53 were elevated while the ratio of Bcl-2 to Bax was declined in miR-342-5p mimic group (miR-342-5p mimic+H2O2). Opposite results were obtained in miR-342-5p inhibitor group (miR-342-5p inhibitor+H2O2). (F) The corresponding grey-scale maps of (E) were shown. *, P<0.05, **, P<0.01, ***, P<0.001. MiR-342-5p, microRNA-342-5p; HUVECs, human umbilical vein endothelial cells; FC, fold change; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.