ccnd1  (MedChemExpress)

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: Identification of CCND1 as a downstream transcript of ZC3H13 via MeRIP‐seq and transcriptomic analyses. (A) The most highly enriched m6A consensus motif identified from MeRIP‐seq data. (B) Density plot showing the distribution of identified m6A peaks across different regions of mRNA transcripts, including the 5′ untranslated region (5′ UTR), coding sequence (CDS), and 3′ untranslated region (3′ UTR). (C) Gene Ontology (GO) biological process enrichment analysis of genes with ZC3H13‐regulated m6A changes. (D and E) The mRNA and protein expression levels of cell cycle‐related genes (CDK1, CCNB1, CCNA2) following ZC3H13 knockdown (sh1, sh2) were assessed by RT‐qPCR (D) and Western blotting (E). (F and G) The expression changes of the aforementioned cell cycle‐related genes after ZC3H13 overexpression (OE) were determined by RT‐qPCR (F) and Western blotting (G). (G) Venn diagram showing the overlap between MeRIP‐seq‐identified candidate m6A‐regulated transcripts and genes enriched in the ZC3H13‐high post‐treatment malignant cell population, identifying 37 potential downstream candidates, including CCND1, CSTB, and NDUFB1. (I‐J) RT‐qPCR (I) and Western blot (J) validation of the suppressive effect of ZC3H13 knockdown on the expression of candidate target genes (CSTB, CCND1, NDUFB1). (K‐L) RT‐qPCR (K) and Western blot (L) validation demonstrating the promoting effect of ZC3H13 overexpression on target gene expression. (M) MeRIP‐qPCR assay quantifying the changes in m6A modification abundance on CCND1 mRNA following ZC3H13 depletion. (N) Integrative Genomics Viewer (IGV) browser tracks displaying the distribution and abundance of sequencing peaks along the CCND1 transcript in the control (ZC3H13) and knockdown (shZC3H13) groups. Data are presented as mean ± SD from three independent biological experiments unless otherwise indicated. * p < .05, ** p < .01, *** p < .00.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Over Expression, Biomarker Discovery, Targeted Gene Expression, Modification, Control

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: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

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

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: Clinical significance of CCND1 in HNSCC and its rescue effect on ZC3H13‐mediated cellular malignant phenotypes. (A) Representative immunohistochemistry (IHC) images demonstrating CCND1 expression in normal oral tissues, anti‐PD‐1 responder tissues, and anti‐PD‐1 non‐responder tumour tissues. (B) Statistical comparison of CCND1 IHC scores between normal and tumour tissues in a clinical cohort of 120 paired samples. (C) Differential analysis of CCND1 protein expression between anti‐PD‐1 responders (n = 38) and anti‐PD‐1 non‐responders ( n = 52) patients. (D) Comparison of CCND1 IHC scores stratified by histological grade (Grade 1–2 vs Grade 3–4). (E) Comparison of CCND1 IHC scores stratified by clinical stage (Stage 1–2 vs. Stage 3–4). (F) Kaplan–Meier survival curves showing the difference in overall survival (OS) between CCND1 high‐expression ( n = 55) and low‐expression ( n = 65) groups. Patients were dichotomized according to the median H‐score of the corresponding marker. (G) Pearson correlation analysis revealing a significant positive correlation between ZC3H13 and CCND1 protein expression (IHC scores) in HNSCC tumour tissues. (H) Western blot analysis of ZC3H13 and CCND1 protein expression levels in cells co‐transfected with Vector, sh_ZC3H13, oe_CCND1, or sh+oe to validate the efficacy of the rescue experiment. (I) CCK‐8 assay evaluating the rescue effect of CCND1 overexpression on the proliferation inhibition induced by ZC3H13 knockdown in HNSCC cells. (J and K) Colony formation assay and quantitative analysis evaluating the restorative effect of CCND1 on the long‐term proliferative capacity of ZC3H13‐depleted cells. (L and M) Transwell migration assay and quantitative analysis confirming that CCND1 reversed the suppressive effect of ZC3H13 knockdown on cell migration in vitro. (N and O) Transwell invasion assay and quantitative analysis verifying that CCND1 rescued the impaired invasive capability of cells induced by ZC3H13 knockdown. Data are presented as the mean ± SD from three independent experiments. * p < .05, ** p < .01, *** p < .001, ns indicates not significant.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Immunohistochemistry, Expressing, Comparison, Marker, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay, Over Expression, Inhibition, Knockdown, Colony Assay, Transwell Migration Assay, Migration, In Vitro, Transwell Invasion Assay

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: The ZC3H13/CCND1 axis remodels the HNSCC immune microenvironment and the proposed mechanistic model. (A) Gene Ontology (GO) enrichment analysis revealing biological pathways related to the negative regulation of cell activation and apoptosis. (B) Bar plot illustrating the changes in the proportion of CD4 + T cells between the Pre‐ and Post‐immunotherapy groups. (C) Boxplot comparing the quantitative CytoTRACE scores before and after immunotherapy (Pre vs. Post). (D) UMAP feature plots displaying the spatial distribution of cell differentiation states (CytoTRACE scores) in Pre‐ and Post‐treatment samples. (E) Violin plot demonstrating the significant difference in CD4+ T cell exhaustion scores between the Pre and Post treatment groups. (F) The relative mRNA expression levels of key immune checkpoint molecules (PDCD1, LAG3, CTLA4) in CCND1‐low and CCND1‐high groups were determined by RT‐qPCR. (G) Representative multiplex immunofluorescence images showing CCND1, CD4, PD‐1 and DAPI staining in mouse HNSCC tissues from control and ZC3H13‐deficient groups. Scale bar, 20 µm. (H) The quantification for the multiplex immunofluorescence (mIF) staining in the tumour microenvironment. (I) Schematic mechanistic model. Data are presented as the mean ± SD. *** p < .001.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Activation Assay, Cell Differentiation, Expressing, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, Control