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: 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