Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: ALKBH5 increases NPC2 expression in the oxaliplatin resistant CRC. ( A , B ) HCT116 and LoVo cells were treated with oxaliplatin (5 µg/mL) for 48 h, then the mRNA level of m 6 A regulators (METTL3, METTL14, METTL16, WATP, ALKBH5, FTO, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2) were measured by RT-qPCR. ( C ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the expression of NPC2 mRNA was measured by RT-qPCR. ( D ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the protein expression of NPC2 and ALKBH5 was measured by Western blot analysis. ( E ) HCT116 cells were transfected with a luciferase reporter construct containing the ALKBH5 promoter, followed by treatment with oxaliplatin (5 µg/mL) for 48 h. Luciferase activity was measured and normalized to Renilla. ( F , G ) HCT116 cells were transfected with ALKBH5 plasmid, then the expression level of ALKBH5 and NPC2 were measured by RT-qPCR ( F ) and Western blot analysis ( G ). ( H ) HCT116 cells were transfected with ALKBH5 plasmid, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( I ) Counting and statistical analysis of the colony numbers. ( J , K ) HCT116 cells were transfected with NC or ALKBH5 siRNA, then the expression level of ALKBH5 and NPC2 were measured by RT-qPCR ( J ) and Western blot analysis ( K ). ( L ) HCT116-OXR cells were transfected with control or ALKBH5-siRNA, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( M ) Counting and statistical analysis of the colony numbers. ( N ) HCT116 cells were transfected with ALKBH5 plasmid, the m 6 A modifications of NPC2 mRNA were measured by MeRIP-qPCR. ( O ) HCT116 cells were transfected with ALKBH5 siRNA, the m 6 A modifications of NPC2 mRNA were measured by MeRIP-qPCR. ( P ) HCT116-OXR cells were treated with oxaliplatin (5 µg/mL), and RIP-PCR was performed to detect the molecular interaction within ALKBH5 and NPC2. ( Q ) The binding capacity between NPC2 mRNA and ALKBH5 protein or YTHDF2 protein in HCT116 cells were examined by RNA pulldown and western blot analysis. ( R ) HCT116 cells were transfected with ALKBH5 plasmid or ALKBH5 siRNA, then treated with ActD (5 µg/mL) for the indicated times. The expression of NPC2 mRNA was examined with RT-qPCR. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The siRNAs targeting NPC2, METTL3, FTO, ALKBH5 and YTHDF2 were purchased from GenePharma.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Colony Assay, Control, Binding Assay

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: YTHDF2 promotes the degradation of NPC2 mRNA in the oxaliplatin resistant CRC. ( A - E ) HCT116 cells were transfected with siRNA of YTHDF1 ( A ), YTHDF2 ( B ), YTHDF3 ( C ), YTHDC1 ( D ), YTHDC2 ( E ), then the mRNA level of YTH domain-containing proteins and NPC2 were measured by RT-qPCR. ( F ) HCT116 cells were transfected with NC or YTHDF2 siRNA, then the protein level of YTHDF2 and NPC2 were measured by Western blot analysis. ( G ) HCT116 cells were transfected with control or YTHDF2-siRNA, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( H ) Counting and statistical analysis of the colony numbers. ( I ) HCT116-OXR cells were treated with oxaliplatin (5 µg/mL), and RIP-PCR was performed to detect the molecular interaction within YTHDF2 and NPC2. ( J ) HCT116 cells were transfected with NC or YTHDF2 siRNA, then treated with ActD (5 µg/mL) for the indicated times. The expression of NPC2 mRNA was examined with RT-qPCR. ( K ) HCT116 cells were transfected with NC or YTHDF2-siRNA then with or without ALKBH5 knockdown. The protein level of ALKBH5, YTHDF2 and NPC2 were measured by Western blot analysis. ( L ) HCT116 cells were transfected with Vector or YTHDF2 plasmid then with or without ALKBH5 overexpression. The protein level of ALKBH5, YTHDF2 and NPC2 were measured by Western blot analysis. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The siRNAs targeting NPC2, METTL3, FTO, ALKBH5 and YTHDF2 were purchased from GenePharma.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Control, Colony Assay, Expressing, Knockdown, Plasmid Preparation, Over Expression

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Role of YTHDF2 in HCC progression and its potential clinical significance. Note: ( A ) Meta-analysis flowchart; ( B ) Forest plot of YTHDF2 in HCC; ( C ) Funnel plot assessing publication bias; ( D ) Sensitivity analysis; ( E ) Differential expression analysis of YTHDF2 between normal and tumor groups based on the TCGA-LIHC dataset (*** p < 0.001); ( F ) Survival curves of high and low YTHDF2 expression groups. Tumor: n = 373; Normal: n = 49

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Molecular mechanism and potential clinical significance of YTHDF2 in HCC. Note: ( A ) Schematic workflow of single-cell microarray analysis; ( B ) Visualization of cell annotation results based on UMAP clustering; ( C ) InferCNV analysis depicting copy number amplification and deletion patterns; ( D ) Visualization of annotated malignant cells based on UMAP clustering; ( E ) UMAP representation of YTHDF2 expression; ( F ) Expression of YTHDF2 in MDSCs across 11 samples; ( G ) Statistical comparison of cancer cell differences between the YTHDF2 high-expression and low-expression groups (* p < 0.05, YTHDF2_Low: n = 7, YTHDF2_High: n = 4)

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Microarray, Amplification, Expressing, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Transcriptomic analysis of MDSCs and MYC screening following YTHDF2 Knockout. Note: ( A ) Workflow of RNA sequencing analysis; ( B ) Volcano plot of DEGs in MDSCs after YTHDF2 knockout, where red indicates significantly upregulated genes, green represents significantly downregulated genes, and gray denotes non-significant genes; ( C ) LASSO coefficient distribution of DEGs; ( D ) Selection of the optimal lambda parameter in the LASSO model; ( E ) Violin plots illustrating the differential expression of CXCL3, SLC45A3, and MYC between the two groups. YTHDF2-WT: n = 3; YTHDF2-KO: n = 3

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Knock-Out, RNA Sequencing, Selection, Quantitative Proteomics

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: YTHDF2 Regulates the Immunosuppressive Function of MDSCs by Modulating MYC and Its m 6 A Modification. Note: ( A ) Schematic illustration of the MYC-mediated regulation of MDSC activity; ( B ) RIP assay showing the binding of YTHDF2 to MYC mRNA; ( C ) meRIP-qPCR assay detecting the m 6 A modification level of MYC mRNA; ( D ) Western blot and RT-qPCR analysis of MYC mRNA and protein expression in MDSCs following YTHDF2 overexpression or knockdown; ( E ) RT-qPCR analysis of iNOS and Arg-1 mRNA expression in MDSCs upon YTHDF2 overexpression or knockdown; ( F ) Schematic diagram of the T cell-MDSC co-culture experiment; ( G ) ELISA analysis of IFN-γ secretion in the supernatant of the co-culture system after YTHDF2 knockdown or overexpression in MDSCs; ( H - J ) Flow cytometry analysis of T cell proliferation in the co-culture system after YTHDF2 knockdown or overexpression in MDSCs. All experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Modification, Activity Assay, Binding Assay, Western Blot, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Construction and characterization of lip@si-YTHDF2. Note: ( A ) Schematic illustration of the preparation of pH-responsive lipid nanoparticles; ( B ) AGE analysis of SA-H8/siRNA complexes at different N/P ratios to evaluate siRNA encapsulation; ( C ) Particle size distribution and zeta potential measured by dynamic light scattering (DLS); ( D ) TEM imaging of lip@si-YTHDF2 morphology; ( E ) AFM analysis of lip@si-YTHDF2 morphology and size distribution; ( F ) siRNA release profile of lip@si-YTHDF2 under different pH conditions; ( G ) Co-localization analysis of Cy5-lip@si-YTHDF2 with early/late endosomes and lysosomes

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Encapsulation, Zeta Potential Analyzer, Imaging

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Impact of lip@si-YTHDF2 Regulation of MYC on MDSCs Function. Note: ( A ) Schematic representation of co-culture experiments between MDSCs and T cells; ( B ) RT-qPCR analysis of YTHDF2 and MYC mRNA expression levels in various MDSC groups; ( C ) Western Blot analysis of YTHDF2 and MYC protein expression levels in various MDSC groups; ( D ) RT-qPCR analysis of iNOS and Arg1 mRNA expression levels in various MDSC groups; ( E - F ) Flow cytometry analysis of T cell proliferation in the co-culture system; ( G ) ELISA quantification of IFN-γ secretion levels in the co-culture supernatants of various groups. * p < 0.05, ** p < 0.01, *** p < 0.001; experiments were performed in triplicate

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Co-Culture Assay, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Effects of lip@si-YTHDF2-mediated MYC regulation on CSC Function. Note: ( A ) Schematic representation of the co-cultivation experiment of MDSCs and CSCs in a Transwell system; ( B ) Proliferation of CSCs under various co-cultivation conditions assessed via CCK-8 assay; ( C ) Migration capabilities of CSCs under various co-cultivation conditions evaluated using scratch assays; ( D ) Invasive potential of CSCs under different co-cultivation conditions assessed through Transwell migration assays; ( E ) mRNA expression levels of stemness-related genes (Nanog, Oct4, and Sox2) and the immune evasion gene (PD-L1) in CSCs, determined by RT-qPCR; ( F ) Protein expression levels of stemness-related genes (Nanog, Oct4, and Sox2) and the immune evasion gene (PD-L1) in CSCs, analyzed by Western Blot. * p < 0.05, ** p < 0.01, *** p < 0.001; experiments were replicated three times

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: CCK-8 Assay, Migration, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Targeting YTHDF2 with pH-responsive siRNA nanoparticles suppresses MYC m6A modification and restores antitumor immunity in hepatocellular carcinoma

doi: 10.1186/s12951-025-03538-0

Figure Lengend Snippet: Assessment of tumor growth and immune response following treatment with lip@si-YTHDF2 combined with PD-1 inhibitor. Note: ( A ) Flowchart of tumor growth following treatment with lip@si-YTHDF2 combined with PD-1 inhibitor; ( B ) The fluorescence signal of tumor-bearing mice was detected using the IVIS system; ( C ) Monitoring results of tumor volume; ( D ) Western blot analysis of YTHDF2, MYC, and stemness genes (Oct4, Nanog, Sox2) expression levels in tumor tissues; ( E ) RT-qPCR analysis of YTHDF2, MYC, and stemness genes (Oct4, Nanog, Sox2) expression levels in tumor tissues. * indicates a significant difference between the groups; *** p < 0.001; n = 6 mice per group

Article Snippet: Synthetic siRNA targeting YTHDF2 (sense: 5’-TTGGCTATTGGGAACGTCCT-3’; antisense: 5’-AGGACGTTCCCAATAGCCAA-3’) was obtained from GenePharma (Shanghai, China) and added to the SA-H8 solution.

Techniques: Fluorescence, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: METTL3-mediated m6A modification regulates D-galactose-induced skin fibroblast senescence through miR-208a-5p

doi: 10.3389/fimmu.2025.1577783

Figure Lengend Snippet: Analysis of METTL3, miR-208a-5p and OPA1 in aging skin. (A) Experimental design overview. (B) Hematoxylin and eosin (H&E) staining of mouse skin tissue after daily subcutaneous injections of D-galactose for three months (n=10). Scale bars: 500μm (overview), 200μm (magnified view). (C) Western blot analysis of P21 expression in aging mice (n=3). (D) qRT-PCR measurement of m6A-modified mRNA levels in aging skin (n=5). (E) Western blot analysis of METTL3 expression in aging skin (n=3). (F) qRT-PCR quantification of miR-208a-5p levels in aging skin (n=3). (G) qRT-PCR analysis of OPA1 mRNA expression in aging skin (n=3). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns was considered statistically insignificant. D-gal: D-galactose. Figure 1A was created with BioRender.com .

Article Snippet: Furthermore, siRNAs targeting METTL3, YTHDF2, and OPA1 (RiboBio, Guangzhou, China), as listed in , were transfected using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Staining, Western Blot, Expressing, Quantitative RT-PCR, Modification

Journal: Frontiers in Immunology

Article Title: METTL3-mediated m6A modification regulates D-galactose-induced skin fibroblast senescence through miR-208a-5p

doi: 10.3389/fimmu.2025.1577783

Figure Lengend Snippet: miR-208a-5p promotes senescence by targeting OPA1. (A) Predicted binding sites of miR-208a-5p on the OPA1 mRNA. (B) A dual luciferase reporter assay confirmed the targeted interaction between OPA1 and miR-208a-5p (n=5). *p < 0.05; ns indicates statistically insignificant. (C) Western blot analysis revealed reduced OPA1 expression 48 hours post-transfection with miR-208a-5p mimic (n=3), **p < 0.01. (D) OPA1 knockdown via siRNA significantly decreased its expression, as shown by Western blot 48 hours post-transfection (n=3), **p < 0.01. (E) SA-β-gal staining quantified cellular senescence in OPA1-deficient MSFs. Scale bars: 100 μm, ***p < 0.001. (F) Mitophagy levels were assessed using a Mitophagy Detection Kit in siNC, siMETTL3, and siMETTL3 + GSK2578215A (GSK) groups. Scale bars: 50μm; *p < 0.05 vs. siNC, ##p < 0.01 vs. siMETTL3; *p < 0.05 vs. NC-mimic, ##p < 0.01 vs. miR-208a-5p mimic. (G) Degree of cellular senescence was measured by SA-β-gal staining (n=3). Scale bars: 100 μm, **p < 0.01 vs. siNC, ###p< 0.001 vs. siMETTL3; ***p < 0.001 vs. NC-mimic, ###p< 0.001 vs. miR-208a-5p mimic. GSK, GSK2578215A.

Article Snippet: Furthermore, siRNAs targeting METTL3, YTHDF2, and OPA1 (RiboBio, Guangzhou, China), as listed in , were transfected using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Binding Assay, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection, Knockdown, Staining