Background: The blood-brain barrier (BBB) strongly restricts the entry of anti-glioma drugs into tumor tissues and thus decreases chemotherapy efficacy. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumor form. Because regulation of the effect of noncoding RNA on BBB function is attracting growing attention, we investigated the effects of noncoding RNA on the permeability of glioma conditioned normal BBB and the mechanism involved using PIWI-associated RNA piR-DQ590027 as a starting point.
Methods: The mRNA levels of MIR17HG, miR-153, miR-377, ZO-1, occludin, and claudin-5 were determined using real-time PCR. Transient cell transfection was performed using Lipofectamine 3000 reagent. TEER and HRP flux were applied to measure the permeability of glioma conditioned normal BBB. Western blotting and immunofluorescence assays were used to measure ZO-1, occludin, and claudin-5 levels. Reporter vector construction and a luciferase reporter assay were performed to detect the binding sites of MIR17HG and piR-DQ590027, MIR17HG and miR-153 (miR-377), and FOXR2 and miR-153 (miR-377). RNA immunoprecipitation was used to test the interaction between miR-153 (miR-377) and its target proteins. Chromatin immunoprecipitation was performed to detect the interaction between the transcription factor FOXR2 and ZO-1, occludin, and claudin-5.
Results: piR-DQ590027 was expressed at low levels in glioma-conditioned ECs (GECs) of the in vitro glioma conditioned normal BBB model. Overexpression of piR-DQ590027 down-regulated the expressions of ZO-1, occludin, and claudin-5 and increased the permeability of glioma conditioned normal BBB. MIR17HG had high expression in GECs but miR-153 and miR-377 had low expression. piR-DQ590027 bound to and negatively regulated MIR17HG. FOXR2 was a downstream target of miR-153 and miR-377; MIR17HG bound separately to miR-153 and miR-377 and negatively regulated their ability to mediate FOXR2 expression. FOXR2 associated with the promoter regions of ZO-1, occludin, and claudin-5 in GECs to promote their transcription.
Conclusion: The piR-DQ590027/MIR17HG/miR-153 (miR-377)/FOXR2 pathway plays an important role in regulating glioma conditioned normal BBB permeability and provides a new target for the comprehensive treatment of glioma.
Keywords: Blood-brain barrier; FOXR2; MIR17HG; miR-153; miR-377; piR-DQ590027.