Fig 2: LINC00673 Was Highly Expressed in GECs, and Knockdown of LINC00673 Increased BTB Permeability and Inhibited the Expression Levels of ZO-1, Occludin, and Claudin-5(A) lncRNA microarray analysis of total RNAs isolated from GECs treated with overexpression of RBFOX1. Red indicates high relative expression, and green indicates low relative expression. qRT-PCR was used to detected the selected molecules. Data represent means ± SD (n = 3, each). ∗p < 0.05, ∗∗p < 0.01 versus the vector group. (B) FISH was conformed to investigate expression and location of LINC00673 in AECs and GECs (red, LINC00673; blue, DAPI nuclear staining). Scale bar represents 20 μm. (C) The expression levels of LINC00673 in AECs and GECs were detected by a qRT-PCR assay. Data represent means ± SD (n = 3, each). ∗p < 0.05 versus the AECs group. (D and E) Effects of LINC00673 upregulation or knockdown on TEER values (D) and HRP flux (E). (F) Effects of LINC00673 upregulation or knockdown on ZO-1, occludin, and claudin-5 expression levels determined by western blot. Data represent means ± SD (n = 3, each). ∗p < 0.05 versus the vector group; #p < 0.05 versus the shNC group. (G) Effects of LINC00673 upregulation or knockdown on ZO-1, occludin, and claudin-5 distribution and expression levels determined by immunofluorescence staining (n = 3, each). ZO-1, occludin, and claudin-5 (red) were labeled with secondary antibodies anti-ZO-1, anti-occludin, and anti-claudin-5, respectively, and nuclei (blue) were labeled with DAPI. Scale bar represents 30 μm.

Fig 3: Upregulation of RBFOX1 Increased BTB Permeability in GECs and Inhibited the Expression Levels of ZO-1, Occludin, and Claudin-5(A) The expression levels of RBFOX1 in glioma and patient survival were analyzed through TCGA database. (B) Staining vessel from glioma or normal brain tissues with UEA-I. LCM capture of UEA-I-stained vessel from human brain sections is shown. Scale bar represents 40 µm. (C) qRT-PCR analysis of RBFOX1 expression in brain microvessels captured by laser capture microdissection (LCM) from a panel of surgical human brain specimens of normal brain tissues (NBTs), low-grade glioma (LGG, WHO I–II), and high-grade glioma (HGG, WHO III–IV). Data represent means ± SD (n = 8, each). *p < 0.05, **p < 0.01 versus the NBTs group; ##p < 0.01 versus the LGG group. (D) The expression levels of RBFOX1 protein in AECs and GECs were detected by Western bolt assay. (E) The expression levels of RBFOX1 mRNA in AECs and GECs were detected by qRT-PCR assay. Data represent means ± SD (n = 3, each). *p < 0.05 versus the AECs group. (F) Effects of RBFOX1 upregulation or knockdown on TEER values. (G) Effects of RBFOX1 upregulation or knockdown on HRP flux. Data represent means ± SD (n = 3, each). *p < 0.05 versus the vector group; #p < 0.05 versus the shNC group. (H) Effect of RBFOX1 upregulation or knockdown on ZO-1, occludin, and claudin-5 protein expression levels as determined by western blot. Data represent means ± SD (n = 3, each). *p < 0.05 versus the vector group; #p < 0.05 versus the shNC group. (I) Effects of RBFOX1 upregulation or knockdown on ZO-1, occludin, and claudin-5 distribution and expression levels as determined by immunofluorescence staining (n = 3, each). ZO-1, occludin, and claudin-5 (red) were labeled with secondary antibodies anti-ZO-1, anti-occludin, and anti-claudin-5, respectively, and nuclei (blue) were labeled with DAPI. Scale bar represents 30 µm.

Fig 4: Upregulation of RBFOX1 Destabilized LINC00673, and LINC00673 Was Involved in RBFOX1-Mediated Regulation on BTB Permeability(A) Effects of RBFOX1 overexpression or inhibition on LINC00673 expression by a qRT-PCR assay. Data represent means ± SD (n = 3, each). *p < 0.05, **p < 0.01 versus the vector group; #p < 0.05, ##p < 0.01 versus the shNC group. (B) Enrichment of LINC00673 in RBFOX1 immunoprecipitates was measured by a RIP assay. Data represent means ± SD (n = 3, each). **p < 0.01 versus the anti-normal IgG group. (C) RBFOX1 and GAPDH protein levels in immunoprecipitation with LINC00673 RNA were evaluated by western blots. The expression levels of RBFOX1 and GAPDH proteins are shown. (D) The Click-iT Nascent RNA capture kit (Life Technologies) was used to label and capture newly synthesized RNA, and nascent LINC00673 was analyzed using qRT-PCR. (E) GECs after RBFOX1 overexpression were exposed to actinomycin D (2 µg/mL), and total RNA was separated at the indicated times and subjected to qRT-PCR to assess the half-life of LINC00673 RNA. Data represent means ± SD (n = 3, each). *p < 0.05 versus the vector group. RBFOX1 and LINC00673 were co-transfected in GECs. (F and G) Effects of RBFOX1 upregulation and LINC00673 knockdown or RBFOX1 knockdown and LINC00673 upregulation on TEER values (F) and HRP flux (G). (H) Effects of RBFOX1 upregulation and LINC00673 knockdown or RBFOX1 knockdown and LINC00673 upregulation on ZO-1, occludin, and claudin-5 expression levels as determined by western blot. Data represent means ± SD (n = 3, each). *p < 0.05, **p < 0.01 versus the RBFOX1 OE+LINC00673 OE group; #p < 0.05, ##p < 0.01 versus the shRBFOX1+shLINC00673 group; ?p < 0.05, ??p < 0.01 versus the control group.