Journal: Genes & Diseases

Article Title: ADAR1p110 promotes hepatocellular carcinoma metastasis via the miR-451a/TUBA1A axis

doi: 10.1016/j.gendis.2025.101770

Figure Lengend Snippet: ADAR1p110 regulates the expression of TUBA1A by inhibiting the expression of miR-451a. (A) Changes of RNA editing ratio after ADAR1p110 overexpression or knockdown. (B) Schematic diagram of TUBA1A editing. (C) Statistics of microRNA editing events in WT and ADAR1p110-overexpressing HCC cells. (D) miRNAs with editing counts greater than 5 and editing ratio greater than 5% in ADAR1p110-overexpressing or ADAR1p110 knockdown cells. (E) miRNAs were down-regulated after ADAR1p110 overexpression and up-regulated after ADAR1 knockdown. (F) The mRNA expression of TUBA1A was negatively correlated with that of miR-451a analysis based on the TCGA-LIHC dataset ( n = 370). (G) The expression level of miR-451a in tumor and non-tumor tissues from the TCGA-LIHC dataset (Tumor n = 369, Non-tumor n = 49). (H) Kaplan–Meier overall survival curves of TCGA-LIHC patients with low or high expressed miR-451a (Low miR-451a n = 248, High miR-451a n = 113). (I) The expression of miR-451a in the indicated cells was verified by qRT‒PCR ( n = 3). (J) The expression of TUBA1A in the indicated cells was verified by qRT‒PCR ( n = 3). (K) The relative expression of miR-451a and TUBA1A in anti-AGO2 antibody precipitated RNA ( n = 3). (L) Schematic diagram of miR-451a binding with the WT and mutated 3′-UTR of TUBA1A. (M) Luciferase activities of TUBA1A-WT or TUBA1A-MUT were determined in the presence of the NC mimic or the miR-451a mimic ( n = 3). The data are presented as the mean ± SD. P values were computed using the unpaired Student's t -test (G, I, J, K, M), one-way ANOVA test (I), Pearson's correlation test (F), and log-rank tests (H). ns: not significant. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: A luciferase assay kit (Yeasen, China) was used to perform the luciferase reporter assay following the manufacturer's protocol.

Techniques: Expressing, Over Expression, Knockdown, Binding Assay, Luciferase

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 functions as a cytoplasmic ceRNA to sequester miR-562 and de-repress COL4A1. (A) Subcellular distribution of circSMAD4 in TC-hMDMs and patient-derived TAMs assessed by nuclear/cytoplasmic fractionation. (B) Representative immunofluorescence/ISH images showing circSMAD4 signals in macrophages (CD163) with nuclear counterstaining (DAPI). Scale bar, 50 μm. (C) Venn diagram of predicted circSMAD4-interacting miRNAs from circInteractome and circBank, yielding a shortlist including miR-562. (D) miR-562 levels following circSMAD4 knockdown in TC-hMDMs. (E–G) pri-miR-562, pre-miR-562, and miR-562 promoter reporter activity after circSMAD4 overexpression. (H) AGO2-RIP enrichment of circSMAD4 and miR-562 relative to IgG in TC-hMDMs. (I) AGO2 immunoblotting after circSMAD4 sense/antisense RNA pull-down in TC-hMDMs. (J) Predicted pairing between miR-562 and circSMAD4 (WT) and the corresponding mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (K) Dual-luciferase assays for circSMAD4-WT/MUT reporters in the presence of miR-562 mimics or inhibitor. (L) Intersection of miRNA target predictions (miRTarBase, miRmap, TargetScan, and miRDB) identifying candidate miR-562 targets. (M) COL4A1 mRNA levels after miR-562 mimics or inhibitor in TC-hMDMs. (N) Predicted miR-562 binding site within the COL4A1 3′UTR (WT) and mutant design. Mutations were introduced within the predicted miR-562 seed-matching region using transition substitutions (A↔G, C↔U) to disrupt miRNA–target pairing while minimizing changes in sequence composition and local RNA structure. (O) Dual-luciferase assays for COL4A1 3′UTR WT/MUT reporters with miR-562 mimics or inhibitor. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Derivative Assay, Fractionation, Immunofluorescence, Knockdown, Activity Assay, Over Expression, Western Blot, Mutagenesis, Sequencing, Luciferase, Binding Assay

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 facilitates IGF2BP2-dependent stabilization of m6A-marked transcripts. (A) Venn diagram intersecting ENCORI-predicted IGF2BP2 targets with DEGs from shIGF2BP2 versus shNC and shcircSMAD4 versus shNC mRNA-seq, identifying shared candidates. (B) MeRIP–qPCR showing m6A enrichment on COL4A1, SPI1, and ACTA2 candidate regions (CRDs) in shNC and shIGF2BP2 cells. (C) IGF2BP2-RIP–qPCR showing IGF2BP2 binding to COL4A1, SPI1, and ACTA2 CRDs in shNC + Vector, shcircSMAD4 + Vector, shNC + IGF2BP2, and shcircSMAD4 + IGF2BP2 groups. (D) Biotin-circSMAD4 pull-down followed by qPCR showing enrichment of COL4A1, SPI1, and ACTA2 CRDs in Vector + shNC, circSMAD4 + shNC, Vector + shIGF2BP2, and circSMAD4 + shIGF2BP2 groups. (E–G) Schematics of m6A-site mutations introduced into COL4A1, SPI1, and ACTA2 reporters. (H–J) Dual-luciferase assays for CRD reporters (WT and m6A-mutant) in Vector, circSMAD4, and IGF2BP2 groups. (K–M) MeRIP–qPCR for WT and m6A-mutant CRD reporters in Vector, circSMAD4, and IGF2BP2 groups. (N–P) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 knockdown with Vector or IGF2BP2 overexpression. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). (Q–S) mRNA decay assays of endogenous COL4A1, SPI1, and ACTA2 following circSMAD4 overexpression with shNC or shIGF2BP2. Half-life estimated by one-phase decay (Y0 = 1, Plateau = 0). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: At 48 h post-transfection, luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Cat# DL101-01), and relative luciferase activity was calculated by normalizing Firefly to Renilla signals.

Techniques: Binding Assay, Plasmid Preparation, Luciferase, Mutagenesis, Knockdown, Over Expression

Journal: Clinical and Translational Medicine

Article Title: ZC3H13‐mediated m6A stabilization of CCND1 promotes malignant progression and is associated with poor anti‐PD‐1 response in HNSCC

doi: 10.1002/ctm2.70750

Figure Lengend Snippet: ZC3H13 enhances CCND1 mRNA stability in an IGF2BP1‐dependent manner. (A and B) Actinomycin D transcription inhibition assay followed by RT‐qPCR to assess the effect of ZC3H13 knockdown (sh) on the degradation rate and stability of CCND1 mRNA in HNSCC cells. (C) Polysome profiling analysis illustrating the distribution of monosomes and polysomes in control (Vector) and ZC3H13‐depleted cells. (D) Schematic illustration of the predicted m6A modification site on the CCND1 transcript and the construction of wild‐type and mutant dual‐luciferase reporter vectors. The mutant reporter was generated by an A‐to‐G substitution within the predicted m6A consensus motif, changing TGCCAG to TGCCGG. (E) Dual‐luciferase reporter assay evaluating the relative luciferase activity of WT or mutant CCND1 reporters following ZC3H13 overexpression (OE). (F) RNA pulldown assay followed by Western blotting to detect the direct binding of candidate m6A reader proteins (YTHDC1, YTHDF2, IGF2BP1, IGF2BP2) to different regions (5' UTR, CDS) of the CCND1 transcript. (G) RIP‐qPCR assay quantifying the specific enrichment of CCND1 mRNA by various m6A reader proteins. (H and I) Actinomycin D RNA decay assay showing the effect of IGF2BP1 knockdown on CCND1 mRNA stability. Accelerated CCND1 mRNA degradation after IGF2BP1 depletion supports IGF2BP1 as a reader protein that stabilizes CCND1 mRNA. Data are presented as mean ± SD from three independent biological experiments. Statistical tests were selected according to the experimental design as described in the section. * p < .05, ** p < .01, *** p < .001.

Article Snippet: At 48 h after transfection, reporter activity was quantified with the MedChemExpress dual‐luciferase kit (HY‐K1013), and Firefly luciferase values were normalized to Renilla luciferase to assess the effect of ZC3H13 on CCND1 regulation.

Techniques: Inhibition, Quantitative RT-PCR, Knockdown, Control, Plasmid Preparation, Modification, Mutagenesis, Luciferase, Generated, Reporter Assay, Activity Assay, Over Expression, Western Blot, Binding Assay