https://orcid.org/0000-0001-7690-0847
Figures & data
Table 1. Primers used for ChIP experiments
Figure 1. Knockdown of IGF2BP2 increased BTB permeability in vitro
(A) Staining vessel from normal or glioma brain tissues with the UEA-I. LCM capture of UEA-I-stained vessel from human brain sections. Scale bars represent 40 μm. (B,C) The mRNA and protein expression levels of IGF2BP2 were higher in glioma tissues than in normal brain tissues (NBTs), and highest in low-grade glioma tissues (LGGTs). Data represent means ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. NBTs group, ##P < 0.01 vs. LGGTs group. (D,E) The mRNA and protein expression levels of IGF2BP2 were higher in glioma endothelial cells (GECs) than in astrocyte-exposed endothelial cells (AECs). Data represent mean ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. AECs group. (F,G) Knockdown of IGF2BP2 decreased TEER values and increased HRP flux. (H) Knockdown of IGF2BP2 decreased the expression levels of ZO-1, occludin and claudin-5. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. IGF2BP2 (+)-NC group, #P < 0.05 vs. IGF2BP2 (-)-NC group. (I) Knockdown of IGF2BP2 damaged the continuity of ZO-1, occludin and claudin-5 distributions. Scale bar represents 30 μm. (J,K) Knockdown of IGF2BP2 decreased the mRNA and protein expression levels of ZNF765. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. IGF2BP2 (+)-NC group, #P < 0.05 vs. IGF2BP2 (-)-NC group. QRT-PCR results were analysed using the relative quantification (2–ΔΔCT) method, and blots were analysed in terms of IDVs. All results were analysed using one-way ANOVA test.
Figure 2. Knockdown of FBXL19-AS1 increased BTB permeability in vitro
(A) LncRNAs microarray analysis of total RNAs from the GECs of silenced IGF2BP2. Red means high expression, green means low expression. QRT-PCR assay was used to evaluated the selected molecules. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. IGF2BP2(-) group. (B) FBXL19-AS1 located in the cytoplasm in AECs and GECs and conspicuously elevated in GECs. Scale bars represent 20 μm. (C) The expression level of FBXL19-AS1 was elevated in GECs that in AECs. Results were analysed using two-tailed paired Student’s t test. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. AECs group. (D,E) Knockdown of FBXL19-AS1 decreased TEER values and increased HRP flux. (F) Knockdown of FBXL19-AS1 decreased the expression levels of ZO-1, occludin and claudin-5. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. FBXL19-AS1 (+)-NC group, #P < 0.05 vs. FBXL19-AS1 (-)-NC group. (G) Knockdown of FBXL19-AS1 damaged the continuity of ZO-1, occludin and claudin-5 distributions. Scale bar represents 30 μm. (H,I) Knockdown of FBXL19-AS1 decreased the mRNA and protein expression levels of ZNF765. Data represent mean ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. FBXL19-AS1 (+)-NC group, #P < 0.05, ##P < 0.01 vs. FBXL19-AS1 (-)-NC group. (J) Knockdown of FBXL19-AS1 increased the half-life time of ZNF765. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. Control group.
Figure3 Upregulation of IGF2BP2 increased FBXL19-AS1 stability, FBXL19-AS1 participated in the process of IGF2BP2 regulating BTB permeability
(A). Knockdown of IGF2BP2 decreased the expression level of FBXL19-AS1. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. IGF2BP2 (+)-NC group, #P < 0.05 vs. IGF2BP2 (-)-NC group. (B) RIP assay evaluated the remarkable enrichment of FBXL19-AS1 in anti-IGF2BP2 immunoprecipitates. Results were analysed using two-tailed paired Student’s t test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. Anti-normal IgG group. (C) IGF2BP2 and GAPDH protein in immunoprecipitation with FBXL19 and FBXL19-AS1 RNA were visualized by western blot. (D) No significant difference of nascent FBXL19-AS1 was found in silenced IGF2BP2 GECs. (E) Knockdown of IGF2BP2 decreased the half-life time of FBXL19-AS1. Results were analysed using two-tailed paired Student’s t test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. IGF2BP2 (-)-NC group. (F,G) Overexpression of FBXL19-AS1 significantly rescued the inhibitory or increasing effects of IGF2BP2 knockdown on TEER value and HRP exudate respectively. (H) Overexpression of FBXL19-AS1 significantly rescued the inhibitory effect of IGF2BP2 knockdown on the expression levels of ZO-1, occludin and claudin-5. (I,J) Overexpression of FBXL19-AS1 significantly rescued the increasing effect of IGF2BP2 knockdown on the mRNA and protein expression levels of ZNF765. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. Control group, ##P < 0.01 vs. IGF2BP2(-)+FBXL19-AS1(+) group, &&P < 0.01 vs. IGF2BP2 (+)+FBXL19-AS1(-) group.
Figure 4. Overexpression of ZNF765 increased BTB permeability in vitro
(A) ZNF765 was mainly located in the cytoplasm in AECs and GECs and conspicuously reduced in GECs. Scale bars represent 20 μm. (B,C) The mRNA and protein expression levels of ZNF765 were decreased in GECs than in AECs. Results were analysed using two-tailed paired Student’s t test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. AECs group. (D,E) Overexpression of ZNF765 decreased TEER values and increased HRP flux. (F,G) Overexpression of ZNF765 decreased the mRNA and protein expression levels of tight junction-associated proteins (TJPs). (H) Overexpression of ZNF765 damaged the continuity of TJPs distributions. Scale bar represents 30 μm. (I-J) Overexpression of ZNF765 decreased the mRNA and protein expression levels of IGF2BP2. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. ZNF765 (+)-NC group, #P < 0.05, ##P < 0.01 vs. ZNF765 (-)-NC group.
Figure 5. FBXL19-AS1 negatively regulated ZNF765 expression through SMD pathway
(A) Dual-luciferase reporter assays were performed to determine the binding sites of FBXL19-AS1 and ZNF765 3ʹ-UTR. Data represent mean ±SD (n = 3, each). *P < 0.05 vs ZNF765 3ʹ-UTR-Wt + FBXL19-AS1 (-)-NC group. (B) Knockdown of FBXL19-AS1 significantly induced the enrichment of FBXL19-AS1 and ZNF765 mRNA with STAU1 respectively. Results were analysed using two-tailed paired Student’s t test respectively. Data represent mean ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. anti-IgG group, #P < 0.05 vs. Control+anti-STAU1 group. (C) Overexpression of FBXL19-AS1, knockdown of STAU1 and knockdown of UPF1 reduced the half-life time of ZNF765 mRNA. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. Control group. (D,E) Knockdown of STAU1 rescued the inhibitory effect of overexpression FBXL19-AS1 on the mRNA and protein expression levels of ZNF765. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. Control group. (F,G) Knockdown of ZNF765 rescued the inhibitory or increasing effect of FBXL19-AS1 knockdown on TEER values or HRP of GECs respectively. (H) Knockdown of ZNF765 rescued the inhibitory effect of FBXL19-AS1 knockdown on the expression of ZO-1, occludin and claudin-5 in GECs. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). **P < 0.01 vs. Control group, ##P < 0.01 vs. FBXL19-AS1 (+)+ZNF765 (+) group, &&P < 0.01 vs. FBXL19-AS1 (-)+ZNF765 (-) group.
Figure 6. ZNF765 bound to ZO-1, occludin, claudin-5 and IGF2BP2 promoters and inhibited their expressions at the transcriptional level
(A-D) Schematic diagrams of different reporter plasmids and relative dual-luciferase activities of ZO-1, occludin, claudin-5 and IGF2BP2. The left part showed the deletion positions on the promoter fragments of ZO-1, occludin, claudin-5 and IGF2BP2 respectively, and the right part showed that the reporter vector activities of them were increased when depleted the putative binding sites of ZNF765. The results were showed after normalized with the co-transfected reference vector (pRL-TK), and relative to the activity of pEX3 empty vector, which the activity was set to 1. Results were analysed using two-tailed paired Student’s t test. Data represent means ± SD (n = 3, each). *P < 0.05, **P < 0.01 vs. empty group. (E,H) Schematic representation of ZO-1, occludin, claudin-5 and IGF2BP2 promoter region 3,000 bps upstream of the transcription start sites (TSS) which were designated as +1. Chromatin Immunoprecipitation PCR products for putative ZNF765 binding sites and an upstream region not supposed to associate with ZNF765 were depicted with bold lines. Dashed arrows represent the primers employed to each PCR. GECs were utilized to conduct ChIP assays. PCR was conducted with the resulting precipitated DNA.
Figure 7. IGF2BP2 knockdown combined with FBXL19-AS1 knockdown increased the effect of DOX in promoting apoptosis of U87 cells
(A) Alone or combination use of IGF2BP2 knockdown and FBXL19-AS1 knockdown increased the promotional effect of DOX on the apoptosis rate of U87 cells. Results were analysed using one-way ANOVA test. Data represent mean ± SD (n = 3, each). *P < 0.05 vs. Control group, #P < 0.05 vs. DOX group, &&P < 0.01 vs. DOX+IGF2BP2 (-) group, ▲▲P < 0.05 vs. DOX+FBXL19-AS1 (-) group. (B) The schematic diagram about the regulation process of IGF2BP2/FBXL19-AS1/ZNF765 axis on BTB permeability.
Table 2. Primers used for qRT-PCR to detect IGF2BP2, FBXL19-AS1 and ZNF765
Supplemental material