Figure 4.
Exosomal microRNAs were not responsible for the ExoHeLa-induced down-regulation of TJ proteins. ExoHeLa was treated with or without triton X100 before being co-incubated with HUVECs for 48 hrs. (a) Protein levels of ZO-1 and CLDN5 of HUVECs were evaluated by western blot. (b) RNA was extracted from exoHeLa and exoHCEC, small RNAs were analysed, and the most abundant small RNAs were listed. (c) Based on the small RNA sequencing results, HUVECs were treated with corresponding microRNA inhibitors along with exoHeLa, then analysed by western blot for protein level of ZO-1 and CLDN5. Quantification of ZO-1 and CLDN5 expression were presented as the percentage of that of PBS group, and the data are shown in a bar graph. (d) Quantification of DICER mRNA in HeLa cells using real-time quantitative PCR after the cells were treated with small interference RNA to knocked down DICER mRNA, (e) Protein level of DICER was examined by western blot. (f) Exosomes secreted by HeLa cells and DICER knocked down (DICER KD) HeLa cells were collected, exosomal RNA was extracted from exoHeLa and exoHeLa DICER KD. MiR1290 and miR3960 levels were analysed by Real-time quantitative PCR. (g) After treated with exoHeLa and exoHeLa DICER KD for indicated duration, HUVECs were subjected to western blot analysis for ZO-1 and CLDN5 proteins. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.