Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 is highly expressed in GBM cell lines and tissues. (A) Heatmap showing hierarchical clustering of 3707 differentially expressed circRNAs between GBM cells and NPCs. (B) Volcano plot showing differentially expressed circRNAs in GBM cells, with 1,965 upregulated and 1,742 downregulated. (C) Venn diagram demonstrating that 45 circRNAs expressed in GBM-derived exosomes overlapped with the 1965 upregulated circRNAs in GBM cells. (D) circRAD18 expression was markedly upregulated in GBM cell-derived exosomes, as identified in the exoRBase database. (E) Schematic representation of the genomic location, circular structure exon composition and back-splice junction of circRAD18. (F) RNase R treatment showing that circRAD18 was resistant to digestion, confirming its circular stability. (G) RT-qPCR products amplified with divergent and convergent primers were analyzed by agarose gel electrophoresis, confirming the circular form of circRAD18. (H) RT-qPCR showed that circRAD18 expression was markedly higher in GBM cell lines than in normal astrocytes. (I) RT-qPCR showing that circRAD18 expression was markedly higher in GBM tissues (n=74) than in adjacent normal brain tissues (n=21). (J) FISH was used to detect the localization of circRAD18 in the cytoplasm. Red denotes circRAD18, while blue shows nuclei stained with DAPI. Scale bar, 20 μ m. Subcellular localization of circRAD18 in (K) U87 and (L) U251 cells was determined by nuclear and cytoplasmic fractionation followed by RT-qPCR. β-actin and U6 were used as cytoplasmic and nuclear markers, respectively. circRAD18 was predominantly localized in the cytoplasmic fraction. All data are presented as mean ± SEM and each experiment was performed in triplicate. ** P<0.01; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; NPCs, neural progenitor cells; RT-qPCR, reverse transcription-quantitative PCR; FISH, fluorescence in situ hybridization.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Staining, Fractionation, Reverse Transcription, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 knockdown suppresses GBM cell proliferation, migration and invasion in vitro . (A) RT-qPCR analysis showing that circRAD18 expression was markedly reduced in U87 and U251 cells after siRNA transfection. (B) RT-qPCR analysis showing that siRNA transfection did not affect the expression levels of linear RAD18 mRNA. CCK-8 assays showing that circRAD18 knockdown markedly inhibited the proliferation of (C) U87 and (D) U251 cells. Wound healing assays showing that circRAD18 knockdown markedly reduced the migration of (E) U87 and (F) U251 cells. Scale bar, 100 μ m. (G) Transwell assays showing that circRAD18 knockdown markedly inhibited the invasion of U87 and U251 cells. Scale bar, 100 μ m. Western blot analysis showing that circRAD18 knockdown reduced the expression of invasion-related proteins MMP2, MMP9, MMP14, N-cadherin and Vimentin in (H) U87 and (I) U251 cells. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Knockdown, Migration, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: CircRAD18 acts as a sponge for miR-1231 in GBM cells. (A) Venn diagram showing miRNA targets of circRAD18 predicted using the CircBank and CircInteractome databases. (B) Volcano plot showing differentially expressed miRNAs in GBM cells, including 373 upregulated and 807 downregulated. (C) Venn diagram illustrating the overlap between predicted miRNAs and downregulated miRNAs from the GSE90603 dataset. (D) Data from the CGGA database showing that miR-1231 expression decreased as glioma grade increased. (E) Using data from the CGGA database, patients were categorized by miR-1231 levels and generated Kaplan-Meier survival curves to compare survival rates between the two groups. (F) FISH was used to detect the co-localization of circRAD18 and miR-1231 in the cytoplasm of GBM cells. circRAD18 is shown in red, miR-1231 in green, and nuclei are stained blue with DAPI. Scale bar, 20 μ m. Nucleocytoplasmic fractionation analysis followed by RT-qPCR demonstrated that miR-1231 was predominantly localized in the cytoplasm of (G) U87 and (H) U251 cells. β-actin and U6 were used as cytoplasmic and nuclear markers, respectively, to validate the fractionation efficiency. (I) Predicted binding sites of miR-1231 in circRAD18, with the Mut version of circRAD18 presented. (J) The dual-luciferase reporter assay determining the binding between circRAD18 and miR-1231. (K) RNA pull-down assay performed to verify the direct binding between circRAD18 and miR-1231. (L) RT-qPCR showed that circRAD18 knockdown markedly increased miR-1231 expression in U87 and U251 cells. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; miRNA, microRNA; CGGA, Chinese Glioma Genome Atlas; FISH, fluorescence in situ hybridization; RT-qPCR, reverse transcription-quantitative PCR; MUT, mutant; WT, wild type.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Expressing, Generated, Staining, Fractionation, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Pull Down Assay, Knockdown, Fluorescence, In Situ Hybridization, Reverse Transcription, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 promotes the proliferation, migration and invasion of GBM cells by sponging miR-1231. CCK-8 assays showing that miR-1231 inhibitor transfection promoted the proliferation of (A) U87 and (B) U251 cells. Wound healing assays showing that miR-1231 inhibitor transfection enhanced the migration of (C) U87 and (D) U251 cells. Scale bar, 100 μ m. (E) Transwell assays showed that miR-1231 inhibitor transfection promoted the invasion of U87 and U251 cells. Scale bar, 100 μ m. CCK-8 assays showing that si-circRAD18 co-transfection attenuated the proliferation-promoting effect of miR-1231 inhibitor in (F) U87 and (G) U251 cells. Wound healing assays showing that si-circRAD18 co-transfection attenuated the migration-promoting effect of miR-1231 inhibitor in (H) U87 and (I) U251 cells. Scale bar, 100 μ m. (J) Transwell assays showing that si-circRAD18 co-transfection attenuated the invasion-promoting effect of miR-1231 inhibitor in U87 and U251 cells. Scale bar, 100 μ m. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; miRNA, microRNA; si, small interfering.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Migration, CCK-8 Assay, Transfection, Cotransfection

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 promotes the proliferation, migration and invasion of GBM cells through the miR-1231/LUC7L2 axis. (A) Western blot analysis showing that circRAD18 knockdown reduced LUC7L2 protein levels in U87 and U251 cells. (B) Western blot analysis showing that miR-1231 inhibitor increased LUC7L2 protein levels in U87 and U251 cells. (C) Western blot analysis showing that si-circRAD18 co-transfection attenuated the increase in LUC7L2 protein levels induced by miR-1231 inhibitor in U87 and U251 cells. (D) Western blot analysis showing that LUC7L2 overexpression increased LUC7L2 protein levels in U87 and U251 cells. (E) RT-qPCR analysis showing that LUC7L2 overexpression increased LUC7L2 expression in U87 and U251 cells. CCK-8 assays showing that LUC7L2 overexpression promoted the proliferation of (F) U87 and (G) U251 cells. Wound healing assays showing that LUC7L2 overexpression enhanced the migration of (H) U87 and (I) U251 cells. Scale bar, 100 μ m. (J) Transwell assays showing that LUC7L2 overexpression promoted the invasion of U87 and U251 cells. Scale bar, 100 μ m. CCK-8 assays showing that circRAD18 knockdown attenuated the proliferation-promoting effect of LUC7L2 overexpression in (K) U87 and (L) U251 cells. Wound healing assays showing that circRAD18 knockdown attenuated the migration-promoting effect of LUC7L2 overexpression in (M) U87 and (N) U251 cells. Scale bar, 100 μ m. (O) Transwell assays showed that circRAD18 knockdown attenuated the invasion-promoting effect of LUC7L2 overexpression in U87 and U251 cells. Scale bar, 100 μ m. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; miRNA, microRNA; si, small interfering; RT-qPCR, reverse transcription-quantitative PCR; OE, overexpressed; NC, negative control.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Migration, Western Blot, Knockdown, Cotransfection, Over Expression, Quantitative RT-PCR, Expressing, CCK-8 Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 knockdown inhibits GBM progression in vivo . (A and B) Representative images of xenograft tumors in nude mice showing that circRAD18 knockdown reduced tumor growth. (C) Tumor growth curves showing that circRAD18 knockdown slowed the growth of subcutaneous xenograft tumors. (D) Tumor volume measurements at day 28 showing that circRAD18 knockdown reduced tumor size compared with the control group. (E) Tumor weights measured on day 28 showing that circRAD18 knockdown decreased tumor mass compared with the control group. (F) RT-qPCR analysis showing that circRAD18 knockdown decreased circRAD18 expression in xenograft tumors. (G) RT-qPCR analysis showing that circRAD18 knockdown increased miR-1231 expression in xenograft tumors. (H) RT-qPCR analysis showing that circRAD18 knockdown decreased LUC7L2 expression in xenograft tumors. (I) IHC staining of xenograft tumors showing that circRAD18 knockdown decreased LUC7L2 protein levels. Scale bar, 100 μ m. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. circ, circular RNA; GBM, glioblastoma; IHC, immunohistochemistry; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Knockdown, In Vivo, Control, Quantitative RT-PCR, Expressing, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: CircRAD18 promotes glioblastoma proliferation, migration and invasion via the miR-1231/LUC7L2 axis

doi: 10.3892/ijmm.2026.5851

Figure Lengend Snippet: circRAD18 is enriched in GBM cell-derived exosomes. (A) Transmission electron microscopy showing the morphology of exosomes derived from HA1800 and U87 cells. Scale bar, 200 nm. (B) NTA showing that exosomes from HA1800 and U87 cells ranged in size from 50-120 nm. (C) Western blot analysis confirmed the presence of exosomal markers CD9 and CD63 in EVs. (D) The expression of circRAD18 in U87 cells and U87 cell-derived exosomes was markedly higher than in HA1800 cells and HA1800 cell-derived exosomes. All data are presented as mean ± SEM and each experiment was performed in triplicate. * P<0.05. circ, circular RNA; GBM, glioblastoma; IHC, immunohistochemistry; RT-qPCR, reverse transcription-quantitative PCR; NTA, nanoparticle tracking analysis; EVs, extracellular vesicles.

Article Snippet: Sangon Biotech Co., Ltd. synthesized the biotin-labeled circRAD18 and NC probes.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Immunohistochemistry, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Horticulture Research

Article Title: PtrbZIP12 improves drought resistance in Populus trichocarpa by directly targeting PtrDHN and PtrPOD

doi: 10.1093/hr/uhag034

Figure Lengend Snippet: PtrbZIP12 directly activates the promoters of PtrDHN and PtrPOD . (a) Relative expression levels of PtrDHN and PtrPOD in PtrbZIP12 -OE versus WT plants. Data are presented as means ± SD from three biological replicates. (b) Verification of the direct regulatory interaction between PtrbZIP12 and the PtrDHN promoter using ChIP-PCR, in which the promoter region (−800 to −1 bp) was divided into four fragments (P1–P4). ‘Input’ represents chromatin before immunoprecipitation, ‘ChIP-’ indicates immunoprecipitation without antibody, and ‘ChIP+’ denotes immunoprecipitation with anti-GFP antibody. (c) Y1H assay demonstrating PtrbZIP12 binding to ABRE motifs; controls included p53-HIS2/pGADT7-Rec2-p53 (positive) and pGADT7-Rec2- PtrbZIP12 /p53-HIS2 (negative). (d and e) Y1H assays showing PtrbZIP12 binding to the promoters of PtrDHN and PtrPOD , with P53-promoter-AUR1-C and AD-Rec-P53 as positive controls, and AD-empty prey vector with AUR1-C driven by the target gene promoter as negative controls. (f) EMSA confirming PtrbZIP12 –ABRE binding; lanes: 1, biotin-labeled probe; 2, labeled probe + PtrbZIP12 protein; 3–5, competition with 10-, 50-, and 100-fold molar excess of unlabeled probe. (g and h) Schematic representation of effector and reporter constructs utilized in the dual-LUC assay, with transient LUC/renillase (REN) coactivation experiments in N. benthamiana leaves demonstrating PtrbZIP12 -mediated activation of PtrDHN (g) and PtrPOD (h).

Article Snippet: Specific biotin-labeled DNA probes targeting the promoter region of the gene were synthesized by Sangon Biotech Co.

Techniques: Expressing, Immunoprecipitation, Y1H Assay, Binding Assay, Plasmid Preparation, Labeling, Construct, Activation Assay