Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: miR-181d suppresses the expression of RAD51 (A) Identification of miR-181d-regulated mRNAs. Venn diagram of mRNA bound to biotinylated (Bi) miR-181d, and mRNA silenced by miR-181d transfection in the A1207 human glioblastoma cell line; n = 3 biological replicates (left). Top 10% mRNAs that scored higher than methyl-guanine methyl transferase (MGMT) in both assays were considered potential downstream effectors of miR-181d. Cross-referencing the miR-181d-bound and -silenced mRNA list yielded 260 candidates. Pathways implicated by these candidate genes are shown (right). (B) Top 10 candidate miR-181d-bound and -silenced mRNAs. Genes involved in DNA repair or homologous recombination are shown in green font. (C) miR-181d suppressed FANCA , FANCC , and RAD51 mRNA expression. mRNA expression was measured using RT-qPCR in miR-181d-transfected or non-targeting miRNA (miR-NT)-transfected LN340 cells. ∗∗ p < 0.01 compared to miR-NT control (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (D) miR-181d suppressed RAD51 protein expression. A representative western blot of RAD51 is shown ( n = 3). LN340 cells were transfected with miR-181d or miR-NT. α-tubulin was used as the protein loading control (upper). Densitometry analysis of RAD51 protein bands (lower). (E) RAD51 mRNA co-precipitated with Bi-miR-181d. Forty-eight hours after Bi-miR-181d or Bi-miR-NT (30 nM) transfection, LN340 cells were lysed and incubated with streptavidin-coated magnetic beads. RT-qPCR assays were performed to determine the relative abundance of RAD51 mRNA bound to the magnetic beads. ∗∗∗ p < 0.001 versus miR-NT control (Student’s t test). Data are presented as the mean ± SD ( n = 5 independent experiments).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: Expressing, Transfection, Homologous Recombination, Quantitative RT-PCR, Control, Western Blot, Incubation, Magnetic Beads

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: Epistasis between RAD51 silencing and miR-181d in ionizing radiation response (A) In vitro epistasis between si RAD51 and miR-181d mimic. Clonogenic survival of CMK3 cells was determined after transfection with siNT, si RAD51 , miR-181d, or combinations of these siRNAs for 24 h, followed by treatment with 0, 3, and 6 Gy of ionizing radiation (IR). ∗∗∗ p < 0.001 versus miR-NT control (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (B) si RAD51 and miR-181d combination enhanced γ-H2AX foci accumulation in response to IR. Representative immunofluorescence images of γ-H2AX foci in CMK3 cells transfected with siNT, si RAD51 , miR-181d, or their combinations exposed to 0, 3, or 6 Gy of IR (upper). Quantification of γ-H2AX foci is provided (lower). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 between indicated groups (Student’s t test). Scale bars are 5 μm. (C) In vivo epistasis between si RAD51 and miR-181d mimic in the patient-derived glioblastoma xenograft CMK3 line. Kaplan-Meier survival curves of mice bearing intracranial CMK3 implants after transfection with the various siRNAs. The mice underwent 3 days of 2 Gy/day radiation starting 7 days after tumor implant ( n = 10 mice/group). (D) Epistasis between si RAD51 and miR-181d mimic in the Direct-Repeat (DR)-GFP HR assay. Top: schematic of the DR-GFP HR assay. Full-length GFP gene was disrupted at the I-SceI-recognition site and separated from the downstream GFP internal repeat. After I-SceI induced a double-strand break (DSB), an HR event involved the utilization of the downstream repeat to generate a functional GFP gene. The percent of GFP + cells is a proxy for this HR event. Bottom: U87MG cells harboring the DR-GFP assay were transfected with si RAD51 , miR-181d mimic, or a combination for 24 h followed by pCBASce transfection to induce DSB at I-SceI site. The percentage of GFP + cells was estimated by flow cytometry and is plotted as a bar graph. ∗∗∗ p < 0.001 versus miR-NT control (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: In Vitro, Transfection, Control, Immunofluorescence, In Vivo, Derivative Assay, Functional Assay, Flow Cytometry

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: Temozolomide-sensitizing effect of miR-181d is reconstituted by silencing of MGMT and RAD51 (A) Reconstitution of the temozolomide (TMZ)-sensitizing effect of miR-181d by silencing of MGMT and RAD51 in vitro . Cells from patient-derived glioblastoma lines (CMK3 and CMK30) were transfected with si NT , si MGMT , si RAD51 , miR-181d, or combinations of these siRNAs for 24 h. The cells were treated with TMZ (100 μM) or DMSO. Viability was assessed by a limiting dilution assay. The fold-increase in the number of cells required for colony formation is plotted as a heatmap. Higher number of cell death (increased red intensity) denotes increased sensitivity. Data are presented as the mean ± SD ( n = 5 independent experiments). Representative images of CMK3 colonies developed after the indicated treatments. (B) Reconstitution of the TMZ-sensitizing effect of miR-181d by silencing of MGMT and RAD51 in vivo . Kaplan-Meier survival curves of nude mice bearing intracranial CMK3 cells transfected with si NT , si MGMT , si RAD51 , miR-181d, or their combinations. The mice were administered TMZ intraperitoneally at 50 mg/kg/day for 5 days after 7 days of tumor implantation. Each group consisted of 5 mice (top). Log-rank test p values for the various comparisons are shown in table form (bottom).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: In Vitro, Derivative Assay, Transfection, Limiting Dilution Assay, In Vivo, Tumor Implantation

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: Temozolomide treatment enhances RAD51 and decreases the steady-state level of miR-181d expression (A) Temozolomide (TMZ) treatment reduces the steady-state level of miR-181d expression. CMK3 cells were treated with 100 μM TMZ or 1% DMSO. The cells were lysed at various time points and subjected to RNA isolation followed by RT-qPCR for miR-181d. ∗∗∗ p < 0.001, ∗∗ p < 0.01 (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (B) TMZ treatment elevates RAD51 mRNA expression. CMK3 cells were transfected with miR-NT or miR-181d for 24 h and treated with 100 μM TMZ or 1% DMSO. The cells were lysed at various time points and subjected to RNA isolation followed by RT-qPCR for RAD51 . ∗∗∗ p < 0.001, ∗∗ p < 0.01 (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (C) RAD51 mRNA expression elevated in TMZ-resistant cells relative to parental CMK3. CMK3 cells were subjected to 500 μM TMZ for 4 weeks, and TMZ-resistant clones were isolated. mRNA was isolated from parental and TMZ-resistant clones and analyzed for RAD51 mRNA by RT-qPCR. The results from representative clones are shown. ∗∗∗ p < 0.001 (Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (D) TMZ-resistant cells showed elevated RAD51 protein expression relative to parental cells. CMK3 parental and resistant cell lysates were subjected to western blotting for RAD51 and α-tubulin protein expression (representative western blots; n = 3 independent experiments) (left). Densitometry analysis of RAD51 protein bands (right). (E) Recurrent clinical glioblastoma specimens express high level of RAD51 relative to newly diagnosed glioblastoma specimens. The Cancer Genome Atlas (TCGA) mRNA expression data of newly diagnosed and recurrent glioblastoma specimens were normalized across the dataset, and RAD51 mRNA expression was plotted ( p = 0.0120). Data are presented as the mean ± SD ( n = 582 glioblastoma specimens). (F) RAD51 expression in matched primary and recurrent glioblastoma tumors from the GLASS Consortium. RNA-seq data from 149 patients with paired treatment-naïve primary (TP) and first-recurrence (R1) glioblastoma samples were analyzed using the GLASS Consortium dataset. Transcript-level counts are summarized to the gene level, low-abundance genes were filtered, and normalized RAD51 expression was compared between TP and R1 tumors. Each line represents a paired sample from an individual patient, illustrating differential RAD51 expression during tumor recurrence.

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Transfection, Clone Assay, Western Blot, RNA Sequencing

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: RAD51 drives acquired resistance to temozolomide (A) Silencing of RAD51 in temozolomide (TMZ)-resistant cells sensitized TMZ activity in vitro . Patient-derived CMK17 parental and TMZ-resistant glioblastoma cells were transfected with si NT , si RAD51 , or miR-181d for 24 h. The cells were treated with TMZ (100 μM) or DMSO. Viability was assessed by a limiting dilution assay. The fold-increase in the number of cells required for colony formation is plotted as a heatmap. Higher number of cell death (increased red intensity) denotes increased sensitivity. Data are presented as the mean ± SD ( n = 5 independent experiments). Representative images of CMK17 parental and resistant clones developed after the indicated treatments. (B) Silencing of RAD51 in TMZ-resistant cells sensitized TMZ activity in vivo . Kaplan-Meier survival curves of nude mice bearing intracranial CMK17 TMZ-resistant cells transfected with si NT, si RAD51 , or miR-181d. The mice were administered TMZ intraperitoneally at 50 mg/kg/day for 5 days after 7 days of tumor implantation ( n = 5 mice/group) (left). Log-rank test p values for the various comparisons are shown in table form (right).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: Activity Assay, In Vitro, Derivative Assay, Transfection, Limiting Dilution Assay, Clone Assay, In Vivo, Tumor Implantation

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: miR-181d sensitized temozolomide-induced radiation resistance (A) Temozolomide (TMZ) treatment-induced homologous recombination (HR) was abolished by RAD51 silencing or miR-181d transfection. Left: schematic of the extrachromosomal HR assay. The assay is based on co-transformation of two plasmids (dl-1 and dl-2) into the CMK3 cells, followed by the isolation of genomic DNA. HR events between these plasmids resulted in a recombinant DNA of 420 bp. Right : CMK3 parental and three independent TMZ-resistant clones (labeled 1, 2, and 3) were transfected with si NT , si RAD51 , miR-181d, or their combination. Twenty-four hours post-transfection, the cells were transfected with dl-1 and dl-2 plasmids. Genomic DNA was isolated after 48 h, and qPCR analysis of the recombinant 420-bp DNA was performed. ∗∗∗ p < 0.001(Student’s t test). Data are presented as the mean ± SD ( n = 3 independent experiments). (B) In vitro TMZ-induced radiation resistance is abolished by miR-181d transfection. CMK3 parental and TMZ-resistant cells were transfected with miR-NT or miR-181d for 24 h, followed by treatment with 0 or 6 Gy ionizing radiation (IR). Clonogenic survival was determined subsequently. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 (Student’s t test). Data are presented as the mean ± SD ( n = 5 independent experiments). (C) miR-181d enhanced γ-H2AX foci accumulation in TMZ-resistant cells in response to IR. Representative immunofluorescence images of γ-H2AX foci in CMK3 parental and resistant cells transfected with miR-NT or miR-181d exposed to 0 or 6 Gy of IR (upper). Quantification of γ-H2AX foci is provided (lower). ∗∗∗ p < 0.001 between indicated groups (Student’s t test). Scale bars are 5 μm. (D) In vivo TMZ-induced radiation resistance is abolished by miR-181d transfection. Kaplan-Meier survival curves of nude mice bearing orthotopic implant of patient-derived glioblastoma xenograft CMK3 (P) or a TMZ-resistant (R) clone isolated after TMZ treatment. The cells were transfected with miR-NT or miR-181d before implant. The mice underwent 5 days of 2 Gy/day IR starting 7 days after tumor implant ( n = 8 mice/group).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: Homologous Recombination, Transfection, Transformation Assay, Isolation, Recombinant, Clone Assay, Labeling, In Vitro, Immunofluorescence, In Vivo, Derivative Assay

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: miR-181d enhances radiation sensitivity and prolongs survival in recurrent glioblastoma (A) Kaplan-Meier survival curves of C57BL/6 mice intracranially implanted with murine GL261-resistant (R) glioblastoma cells (clones of GL261 selected for TMZ resistance; see ) and injected with either miR-NT or miR-181d, followed by ionizing radiation (IR) or sham treatment. (B) Log-rank test p values for the various comparisons are shown in table form. (C) GL261R recurrent glioblastoma cells were implanted into the previously survived mice (from A) and injected with miR-NT or miR-181d, with or without IR. Tumor burden of the mice was analyzed by bioluminescence imaging. ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 between indicated groups (Student’s t test).

Article Snippet: For the murine recurrent glioblastoma, GL261 recurrent glioblastoma cells were injected into the brains of an athymic nude mice at 6-weeks of age (Charles River Laboratories, MA).

Techniques: Clone Assay, Injection, Imaging