Abstract
Blood–tumor barrier (BTB) presents a major obstacle to brain drug delivery. Therefore, it is urgent to enhance BTB permeability for the treatment of glioma. In this study, we demonstrated that MIAT, ZAK, and phosphorylated NFκB-p65 (p-NFκB-p65) were upregulated, while miR-140-3p was downregulated in glioma-exposed endothelial cells (GECs) of BTB compared with those in endothelial cells cocultured with astrocytes (ECs) of blood–brain barrier (BBB). MIAT inhibited miR-140-3p expression, increased the expression of ZAK, enhanced the ratio of p-NFκB-p65:NFκB-p65, and promoted the endothelial leakage of BTB. Our current study revealed that miR-140-3p was complementary to the ZAK 3′untranslated regions (3′-UTR), and luciferase activity of ZAK was inhibited by miR-140-3p in 293T cells. MiR-140-3p silencing resulted in an increase in BTB permeability by targeting ZAK, while overexpression of miR-140-3p had the opposite results in GECs of BTB. Overexpression of ZAK induced an increase in BTB permeability, and this effect was related to ZAK’s ability to mediate phosphorylation of NFκB-p65. Conversely, ZAK silencing get opposite results in GECs of BTB. As a molecular sponge of miR-140-3p, MIAT attenuated its negative regulation of the target gene ZAK by adsorbing miR-140-3p. P-NFκB-p65 as a transcription factor negatively regulated the expression of TJ-associated proteins by means of chip assay and luciferase assay. Single or combined application of MIAT and miR-140-3p effectively promoted antitumor drug doxorubicin (Dox) across BTB to induce apoptosis of glioma cells. In summary, MIAT functioned as a miR-140-3p sponge to regulate the expression of its target gene ZAK, which contribution to phosphorylation of NFκB-p65 was associated with an increase in BTB permeability by down-regulating the expression of TJ associated proteins, thereby promoting Dox delivery across BTB. These results might provide a novel strategy and target for chemotherapy of glioma.
Highlights
Glioma originating from human neuroectoderm is a most common type of intracranial tumor in central nervous system (CNS) which accounts for more than 50% in the CNS tumor, showing high incidence and low survivalThe blood–tumor barrier (BTB) locating between brain tumor cells and microvessels can severely block the entry of macromolecules from the blood into tumors, resultingOfficial journal of the Cell Death Differentiation AssociationHe et al Cell Death and Disease (2020)11:936 in poor prognosis and low efficacy of glioma[2,3]
In this study, we demonstrated that MIAT was highly expressed in glioma-exposed endothelial cells (GECs), and the increased expression of MIAT enhanced the permeability of BTB
We provided direct evidence that MIAT was involved in modulating BTB permeability
Summary
Reporter vectors construction and luciferase assays HEK-293T cells were seeded in a 96-well plate. ZO-1-5′UTR, occludin-5′UTR and claudin-5-5′UTR were amplified by PCR and cloned into the pmirGLO Dual Luciferase Expression Vector (Promega, Madison, WI, USA) to construct wild-type and mutation-type luciferase reporter vectors (Generay Biotech Co., Shanghai, China). The potential binding sequence and the corresponding mutant sequence of miR-140-3p in MIAT, were amplified by PCR and cloned into the pmirGLO DualLuciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to construct wild-type and mutation-type luciferase reporter vectors (Generay Biotech Co., Shanghai, China). The biological software Targetscan program (http://www.targetscan.org/) predicted the possible target genes and binding sequences of miR-140-3p, chemically synthesized the wild-type ZAK-WT and mutant ZAKMUT, and cloned the dual luciferase reporter plasmid containing the pmirGLO promoter.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
