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. Author manuscript; available in PMC: 2026 Jan 6.
Published in final edited form as: Cell Rep. 2025 Nov 10;44(11):116516. doi: 10.1016/j.celrep.2025.116516

Figure 4. miR-181d regulates PNPT1 expression.

Figure 4.

(A) miR-181d miRNA response elements (MREs) in PNPT1 3′ UTR. Pink block: predicted MREs of miR-181d (MRE1: 705–729, MRE2: 1250–1274, and MRE3: 1464–1487 bp). miR-181d binding sites within the MREs and mutated-MREs that disrupted miR-181d are depicted below the schema.

(B) CMK3 cells were treated with TMZ or DMSO. Total RNA was extracted, and PNPT1 expression was analyzed using RT-qPCR. GAPDH was used as the control.

(C) PNPT1 protein expression by western blotting in CMK3 cells used in (B).

(D) PNPT1 mRNA expression was quantified in 10 matched pairs of newly diagnosed and recurrent glioblastoma specimens using RT-qPCR.

(E and F) PNPT1 mRNA (E) and PNPT1 protein expressions (F) were assessed after CMK3 cells were transfected with miR-181d or miR-NT mimic. **p < 0.01 indicates statistically significant differences compared to miR-NT.

(G) CMK3 cells transfected with anti-miR-181d or anti-miR-NT. The cell lysate was analyzed for PNPT1 expression by western blotting.

(H) CMK3 cells transfected with Bi-miR-181d or -NT mimic. The lysate was affinity-purified with streptavidin-magnetic beads. Isolated RNA was analyzed for PNPT1 by RT-qPCR. Statistical significance **p < 0.01.

(I) Luciferase reporter assay of miR-181d MREs and its mutants in PNPT1 3′ UTR with miR-181d or -NT mimic. Statistical significance *p < 0.05. Data are represented as mean ± SD.