Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Hsa_circ_0044097 Serves as a Promising Biomarker of Atherosclerosis and Its Effects on Vascular Smooth Cell Proliferation and Migration

doi: 10.1002/jbt.71031

Figure Lengend Snippet: Hsa_circ_0044097 affects the proliferation and migration of HASMCs. (A) RT‐qPCR was used to verify the transfection effect of pcDNA3.1‐hsa_circ_0044097. (B) Overexpression of hsa_circ_0044097 inhibits the levels of IL‐6 and TNF‐α in HASMCs induced by ox‐LDL. (C) Overexpression of hsa_circ_0044097 inhibited the cell proliferation of HASMCs induced by ox‐LDL. (D) Overexpression of hsa_circ_0044097 inhibited PCNA and Cyclin D1 expression in HASMCs cells induced by ox‐LDL. (E) Overexpression of hsa_circ_0044097 inhibited the cell migration of HASMCs induced by ox‐LDL. (F) Overexpression of hsa_circ_0044097 inhibited MMP‐9 and OPN expression in HASMCs cells induced by ox‐LDL. ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with non‐fat milk, the membranes were incubated overnight at 4°C with primary antibodies against PCNA (1:1000, HY‐ P80268 , MCE, Shanghai, China), Cyclin D1 (1:500, HY‐ P80633 , MCE, Shanghai, China), MMP9 (1:500, HY‐ P80756 , MCE, Shanghai, China), osteopontin (OPN) (1:500, HY‐ P86670 , MCE, Shanghai, China) and GAPDH (1:10000, HY‐P80137, MCE, Shanghai, China).

Techniques: Migration, Quantitative RT-PCR, Transfection, Over Expression, Expressing

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Hsa_circ_0044097 Serves as a Promising Biomarker of Atherosclerosis and Its Effects on Vascular Smooth Cell Proliferation and Migration

doi: 10.1002/jbt.71031

Figure Lengend Snippet: Hsa_circ_0044097 affects the proliferation and migration of HASMCs by miR‐3918. (A) MiR‐3918 expression was upregulated in AS patients. (B) The effect of transfection with miR‐3918 mimics/inhibitors on the luciferase activity of circ‐WT and circ‐MUT. (C) The RIP experiment verified the interaction between hsa_circ_0044097 and miR‐3918 in cells. (D) The Spearman correlation analysis for hsa_circ_0044097 and miR‐3918. (E) The transfection effects of pcDNA3.1‐hsa_circ_0044097 and miR‐3918 mimics were verified by RT‐qPCR. (F) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the inflammatory factors (IL‐6 and TNF‐α) induced by ox‐LDL in HASMCs. (G) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the proliferation induced by ox‐LDL in HASMCs. (H) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the PCNA and Cyclin D1 expression induced by ox‐LDL in HASMCs. (I) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the migration induced by ox‐LDL in HASMCs. (J) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the MMP‐9 and OPN expression induced by ox‐LDL in HASMCs. *** p < 0.001.

Article Snippet: After blocking with non‐fat milk, the membranes were incubated overnight at 4°C with primary antibodies against PCNA (1:1000, HY‐ P80268 , MCE, Shanghai, China), Cyclin D1 (1:500, HY‐ P80633 , MCE, Shanghai, China), MMP9 (1:500, HY‐ P80756 , MCE, Shanghai, China), osteopontin (OPN) (1:500, HY‐ P86670 , MCE, Shanghai, China) and GAPDH (1:10000, HY‐P80137, MCE, Shanghai, China).

Techniques: Migration, Expressing, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Hsa_circ_0044097 Serves as a Promising Biomarker of Atherosclerosis and Its Effects on Vascular Smooth Cell Proliferation and Migration

doi: 10.1002/jbt.71031

Figure Lengend Snippet: Hsa_circ_0044097 affects the proliferation and migration of HASMCs through the miR‐3918/CBS axis. (A) Venn diagram of the downstream target genes of miR‐3918. (B) CBS expression was downregulated in AS patients. (C) The effect of transfection with miR‐3918 mimics/inhibitors on the luciferase activity of CBS‐WT and CBS‐MUT. (D) The RIP experiment verified the interaction between CBS and miR‐3918 in cells. (E) The Spearman correlation analysis for CBS and miR‐3918. (F) The transfection effects of pcDNA3.1‐hsa_circ_0044097, miR‐3918 mimics, and pcDNA3.1‐CBS were verified by RT‐qPCR. (G) The ELISA kit measured the levels of inflammatory factors (IL‐6 and TNF‐α). (H) The CCK‐8 method evaluated the proliferation ability of cells. (I) Western blot was used to detect the protein expression levels of PCNA and Cyclin D1. (J) The Transwell method evaluated the migration ability of cells. (K) Western blot was used to detect the protein expression levels of MMP‐9 and OPN. *** p < 0.001.

Article Snippet: After blocking with non‐fat milk, the membranes were incubated overnight at 4°C with primary antibodies against PCNA (1:1000, HY‐ P80268 , MCE, Shanghai, China), Cyclin D1 (1:500, HY‐ P80633 , MCE, Shanghai, China), MMP9 (1:500, HY‐ P80756 , MCE, Shanghai, China), osteopontin (OPN) (1:500, HY‐ P86670 , MCE, Shanghai, China) and GAPDH (1:10000, HY‐P80137, MCE, Shanghai, China).

Techniques: Migration, Expressing, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Western Blot

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

Journal: Anti-Cancer Drugs

Article Title: Hematopoietic progenitor kinase 1 inhibitor BGB-15025 induces apoptosis in acute myeloid leukemia cells through the cell cycle pathway and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway signaling axis

doi: 10.1097/CAD.0000000000001794

Figure Lengend Snippet: BGB-15025 inhibits the cell cycle and the MAPK/ERK signaling pathway in AML cells. (a) KEGG analysis revealed that differentially expressed genes were significantly enriched in relevant signaling pathways. (b) GSEA of differentially expressed genes in the treated group, compared with the control group, indicated a predominant enrichment in cell cycle-related pathways. (c) Two AML cell lines (KG1A and THP-1) were exposed to different concentrations of BGB-15025, and the expression levels of CCND1 , CDK4 , and P21 genes were quantified using qRT-PCR. (d) Various concentrations of BGB-15025 were administered to two AML cell lines (KG1A and THP-1), followed by the detection of cyclin D1, CDK4, and P21 protein expressions via Western blot analysis. (f) Different concentrations of BGB-15025 were administered to two AML cell lines, KG1A and THP-1. The expression levels of ERK, p-ERK, P38, and p-P38 proteins were assessed using Western blot analysis. Data presented are derived from at least three independent experiments. Statistical significance was determined as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 when compared with the control group. (e and g) The effect of HPK1 knockdown on the expression of the above-mentioned proteins was assessed in AML (THP-1) cells. AML, acute myeloid leukemia; GSEA, Gene Set Enrichment Analysis; HPK1, hematopoietic progenitor kinase 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; qRT-PCR, quantitative real-time PCR.

Article Snippet: Membranes were subsequently incubated overnight at 4 °C with specific primary antibodies: β-actin (#4970; 1 : 1000), HPK1 (#46510; 1 : 1000), cyclin D1 (#55506; 1 : 1000), P21 (#2947; 1 : 1000), ERK (#4696; 1 : 1000), phosphorylated ERK (p-ERK, #4370; 1 : 1000), P38 MAPK (#8690; 1 : 1000), and phosphorylated P38 MAPK (p-P38, #9211; 1 : 1000) (all from Cell Signaling Technology, Danvers, Massachusetts, USA).

Techniques: Protein-Protein interactions, Control, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Knockdown, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: m 6 A in adipose tissue inflammation: A novel regulator of obesity and metabolic diseases (Review)

doi: 10.3892/ijmm.2026.5795

Figure Lengend Snippet: Role of m 6 A in adipogenesis. Insufficient adipogenesis in adipose tissue leads to persistent, chronic inflammation. m 6 A modification plays a crucial role in all stages of adipogenesis, from commitment to terminal differentiation. During commitment, METTL3 promotes lipogenic differentiation in BMSCs by regulating the m 6 A levels of PTH1R and JAK1, whereas silencing METTL14 reduces the expression of SMAD1, inhibiting BMSC proliferation. During terminal differentiation, m 6 A regulates MCE and the transition to mature adipocytes. FTO influences key genes such as ATG5, ATG7 and JAK2, affecting autophagy, STAT3 phosphorylation and adipogenesis. FTO knockout increases the m 6 A levels of CCND1 and CDK2, blocking MCE. m 6 A, N6-methyladenine; METTL, methyltransferase-like; PTH1R, parathyroid hormone 1 receptor; JAK, Janus kinase; BMSC, bone marrow mesenchymal stem cell; MCE, mitotic clone amplification; FTO, Fat mass and obesity-associated protein; ATG, autophagy-related; STAT3, signal transducer and activator of transcription 3; CCND1, cyclin D1; CDK2, cyclin-dependent kinase 2; IGF2BP1, insulin-like growth factor 2 mRNA-binding protein 1; YTHDF2, YTH domain family 2.

Article Snippet: In addition, for mitotic clone amplification (MCE) in the early stage of terminal differentiation, the inhibition of FTO expression in 3T3-L1 cells leads to increased m 6 A methylation levels of cyclin D1 (CCND1) and cyclin-dependent kinase 2, the protein expression of which is reduced after recognition by YTHDF2, resulting in blockade of the MCE process and in turn the inhibition of lipogenesis ( ) ( ).

Techniques: Modification, Expressing, Phospho-proteomics, Knock-Out, Blocking Assay, Amplification, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: m 6 A in adipose tissue inflammation: A novel regulator of obesity and metabolic diseases (Review)

doi: 10.3892/ijmm.2026.5795

Figure Lengend Snippet: Role of m 6 A in ATMs. ATMs are deeply involved in adipose tissue inflammation, and m 6 A plays critical roles in macrophage biology, including their development, activation, pyroptosis and metabolism of lipids. (A) m 6 A regulates macrophage development by targeting genes such as CCND1 and ATRX via YTHDF3, ALKBH5 and METTL3, affecting haematopoietic stem and progenitor cell differentiation. (B) m 6 A modification mediated by METTL3, METTL14 and IGF2BP2 controls macrophage activation and polarization by influencing key genes such as SPRED2, MYD88 and STAT1, which impact the NF-κB and PPAR-γ pathways. (C) m 6 A regulates macrophage pyroptosis by targeting CASPASE-1, IL-1β and MALAT1 and modulating pathways such as the PTBP1/USP8/TAK1 pathway. (D) Additionally, m 6 A affects macrophage lipid metabolism by regulating lipid uptake and cholesterol efflux through MSR1 and SR-B1. m 6 A, N6-methyladenine; ATMs, adipose tissue macrophages; CCND1, cyclin D1; ATRX, α-thalassemia X-linked intellectual disability syndrome; YTHDF3, YTH domain family 3; ALKBH5, alkB homologue 5; METTL, methyltransferase-like; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; SPRED2, sprouty-related EVH1 domain-2; MYD88, myeloid differentiation primary response 88; STAT1, signal transducer and activator of transcription 1; NF-κB, nuclear factor-κB; PPAR-γ, peroxisome proliferator-activated receptor γ; CASPASE-1, cysteinyl aspartate specific proteinase-1; IL, interleukin; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; PTBP1, polypyrimidine tract-binding protein 1; USP8, ubiquitin-specific peptidase 8; TAK1, TGFβ-activated kinase 1; MSR1, macrophage scavenger receptor 1; SR-B1, scavenger receptor type B1; ROS, reactive oxygen species; TSC1, tuberous sclerosis complex 1; SOCS2, suppressor of cytokine signalling 2; GSDMD-N, gasdermin D N-terminal domain; OxLDL, oxidized low-density lipoprotein; MSR1, macrophage scavenger receptor 1; DDX5, DEAD-box helicase 5; MEHP, mono(2-ethylhexyl) phthalate.

Article Snippet: In addition, for mitotic clone amplification (MCE) in the early stage of terminal differentiation, the inhibition of FTO expression in 3T3-L1 cells leads to increased m 6 A methylation levels of cyclin D1 (CCND1) and cyclin-dependent kinase 2, the protein expression of which is reduced after recognition by YTHDF2, resulting in blockade of the MCE process and in turn the inhibition of lipogenesis ( ) ( ).

Techniques: Activation Assay, Cell Differentiation, Modification, Binding Assay, Ubiquitin Proteomics