Abstract
The primary goals of this study were to investigate the potential roles of miR-200b in regulating RMP7-induced increases in blood-tumor barrier (BTB) permeability and some of the possible molecular mechanisms associated with this effect. Microarray analysis revealed 34 significantly deregulated miRNAs including miR-200b in the BTB as induced by RMP7 and 8 significantly up-regulated miRNAs in the BTB by RMP7. RMP7 induced tight junction (TJ) opening of the BTB, thereby increasing BTB permeability. Associated with this effect of RMP7 was a decrease in miR-200b expression within the human cerebral microvascular endothelial cells line hCMEC/D3 (ECs) of the BTB. Overexpression of miR-200b inhibited endothelial leakage and restored normal transendothelial electric resistance values. A simultaneous shift in occludin and claudin-5 distributions from insoluble to soluble fractions were observed to be significantly reduced. In addition, overexpression of miR-200b inhibited the relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as the production of stress fiber formation in GECs (ECs with U87 glioma cells co-culturing) of the BTB. MiR-200b silencing produced opposite results as that obtained from that of the miR-200b overexpression group. Overexpression of miR-200b was also associated with a down-regulation in RhoA and ROCKII expression, concomitant with a decrease in BTB permeability. Again, results which were opposite to that obtained with the miR-200b silencing group. We further found that miR-200b regulated BTB permeability by directly targeting RhoA and ROCKII. Collectively, these results suggest that miR-200b's contribution to the RMP7-induced increase in BTB permeability was associated with stress fiber formation and TJ disassembly as achieved by directly targeting RhoA and ROCKII.
Highlights
RMP7, a synthetic bradykinin (BK) analog, is a potent and specific BK B2 receptor agonist
Microarray analysis revealed 34 significantly deregulated miRNAs including miR-200b in the blood-tumor barrier (BTB) as induced by RMP7 and 8 significantly up-regulated miRNAs in the BTB by RMP7 as compared with that observed within the BTB control (Figure 1B)
Use of gene chip technology failed to detect expression changes of miR-181a and miR-34c in glioma endothelial cells (GECs) of the BTB by RMP7. These results suggest that miR-181a and miR-34c are not involved in mediating RMP7-induced increases in BTB permeability
Summary
RMP7, a synthetic bradykinin (BK) analog, is a potent and specific BK B2 receptor agonist. The interaction of BK or RMP7 with the B2 receptor results in a rapid, but transient, increase in the permeability of the blood-brain barrier (BBB) and blood-tumor barrier (BTB) It has been reported that intracarotid infusion of RMP-7 is shown to increase the delivery of carboplatin more than 2-fold and to improve survival in a rat glioma model (Emerich et al, 1999). In line with these findings, the results from our laboratory demonstrated that BK increased the permeability of the BTB (Ma and Xue, 2010; Ma et al, 2012). The paucity of data on miRNA alterations during the process of RMP7-induced TJ opening of the BTB and the implications for the potential use of miRNAs as biomarkers in therapeutic approaches led us to evaluate the differential expression of miRNAs by microarray analysis and quantitative real-time PCR (Quantitative RT-PCR) in response to RMP7
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