ABSTRACT Mitophagy plays a key role in glioma radioresistance, but its upstream regulatory mechanism is not fully understood. This study aims to investigate the function and potential molecular mechanism of polypyrimidine tract‐binding protein 1 (PTBP1) in mitophagy and radioresistance of glioma. The GSE16011 dataset was used to screen the differentially expressed genes, combined with weighted gene co‐expression network analysis and machine learning to screen the key genes related to mitophagy in glioma. The expression levels of PTBP1, ubiquitin‐specific protease 4 (USP4) and prohibitin 2 (PHB2) were detected by qRT‐PCR and western blot. After transfection of overexpression vectors and shRNAs, EdU, flow cytometry, JC‐1 staining, MitoSOX staining, and clonogenic survival assays were used to evaluate cell proliferation, apoptosis, mitochondrial membrane potential, ROS level, and radiosensitivity under 8 Gy irradiation. The interaction between PTBP1 and PHB2 mRNA was verified by RIP, dual‐luciferase reporter assay and actinomycin D assay. Co‐IP, ubiquitination assay and cycloheximide experiment were used to verify the deubiquitination regulation of USP4 on PTBP1. The effect of USP4/PTBP1 axis on the radioresistance of glioma in vivo was verified by constructing a subcutaneous xenograft tumor model combined with 10 Gy irradiation. Bioinformatics analysis identified PTBP1 as a key gene related to mitophagy in glioma, which was highly expressed in glioma tissues and cells. PTBP1 overexpression promoted glioma cell proliferation, inhibited apoptosis, enhanced mitophagy, maintained mitochondrial membrane potential, reduced mitochondrial ROS level, and promoted radioresistance under the irradiation conditions. These effects were reversed by mitophagy inhibitor BafA1. Mechanistically, PTBP1 directly bound to the 3'UTR of PHB2 to enhance its mRNA stability and expression. Overexpression of PHB2 reversed the inhibitory effect of PTBP1 knockdown on mitophagy and radioresistance. Moreover, USP4 inhibited the degradation of PTBP1 by deubiquitination to stabilize its protein level. USP4 knockdown inhibited mitophagy and radioresistance, while PTBP1 overexpression reversed these effects. In vivo experiments further confirmed that USP4 overexpression promoted glioma radioresistance and tumor growth, while PTBP1 knockdown abolished these effects. USP4 stabilizes PTBP1 protein to enhance PHB2 mRNA stability, thereby promoting mitophagy and driving glioma radioresistance. Targeting the USP4/PTBP1/PHB2 axis may represent a potential strategy to sensitize glioma cells to radiation, although this requires further validation with appropriate 0 Gy controls and direct mitophagic flux assays.
ABSTRACT To investigate the biological functions of PPP1R14B in lung adenocarcinoma and further elucidate its underlying regulatory mechanisms. The Cancer Genome Atlas clinical cohorts, single‐cell sequencing, Kaplan–Meier survival analysis, immunohistochemistry, qRT‐PCR, and Western blot were performed to assess the expression and prognostic value of PPP1R14B in lung adenocarcinoma. Then, PPP1R14B‐silenced A549 cells and PPP1R14B‐overexpressing H1650 cells were constructed. Colony formation, CCK‐8, EdU assay, flow cytometry, scratch, and Transwell chamber assays were utilized to evaluate cell proliferation, apoptosis, migration, invasion, and paclitaxel (PTX) resistance. To elucidate the molecular mechanism of PPP1R14B, transcriptome sequencing (RNA‐seq) and co‐immunoprecipitation assays were conducted. Additionally, a xenograft tumor mouse model was established to explore the in vivo role of PPP1R14B. PPP1R14B exhibited high expression in lung adenocarcinoma and correlated with an unfavorable prognosis. Silencing of PPP1R14B markedly suppressed the proliferation, migration, and invasion of A549 cells, while concurrently promoting apoptosis and sensitizing cells to PTX. Nevertheless, overexpression of PPP1R14B in H1650 cells displayed an opposite trend. PPP1R14B directly interacted with ARPC1A and promoted its expression. Overexpression of ARPC1A reversed the inhibitory effects of PPP1R14B silencing on the malignant biological behaviors of A549 cells. In vivo, silencing of PPP1R14B repressed tumor growth, as evidenced by smaller tumor volume, lower tumor weight, fewer Ki67‐positive cells, and more TUNEL‐positive cells. PPP1R14B directly binds ARPC1A and increases its expression to facilitate lung adenocarcinoma tumorigenesis and PTX resistance.
ABSTRACT Recent studies have demonstrated that RING1 and YY1 Binding Protein (RYBP) participates in cell cycle arrest and apoptosis regulation, yet the mechanism by which RYBP is regulated in hepatocellular carcinoma (HCC) remains poorly defined. In this study, we detected the expression levels of RYBP and DNMT3a in human HCC cell lines and the immortalized normal liver cell line THLE‐3 using quantitative real‐time PCR (qPCR) and Western blot. Methylation‐specific PCR (MSP), dual‐luciferase reporter assays and ChIP‐qPCR were performed to investigate the interaction between DNMT3a and the RYBP promoter. EdU incorporation, CCK‐8 and flow cytometry assays were adopted to assess cell proliferation and apoptosis. We found that RYBP was markedly downregulated in HCC cell lines. Ectopic overexpression of RYBP inhibited proliferation, triggered G1‐phase cell cycle arrest and facilitated apoptosis in HCC cells. DNMT3a was overexpressed in HCC cell lines and repressed RYBP expression through promoter methylation. Knockdown of DNMT3a reduced methylation of the RYBP promoter, restored RYBP expression, suppressed cell proliferation and induced apoptosis in HCC cells. Furthermore, RYBP silencing reversed the biological phenotypes induced by DNMT3a knockdown. Collectively, our findings reveal that DNMT3a modulates cell proliferation and apoptosis in HCC via epigenetic repression of RYBP by mediating RYBP promoter methylation.
ABSTRACT RNA‐binding protein RBM4 regulates post‐transcriptional programs, but its role in shaping the hepatocellular carcinoma (HCC) tumor immune microenvironment (TIME) is unclear. We hypothesized that RBM4 enhances S100A10 mRNA stability to drive immune evasion. RBM4–S100A10 coupling was interrogated by expression profiling, RIP‐qPCR, actinomycin D chase, and 3′UTR reporter assays. Gain/loss models (OE‐RBM4, shS100A10) were used to assess migration/invasion and apoptosis. Immune consequences were evaluated by CD8 + T‐cell co‐culture (ELISA for Granzyme B, IFN‐γ, TNF‐α; IL‐10, TGF‐β) and immunofluorescence. Western blots probed cPLA2/5‐LOX signaling. Subcutaneous/orthotopic models measured tumor growth, CD8 + infiltration, and effector subsets; S100A10 silencing and PD‐1 blockade served as interventions. RBM4 positively correlated with S100A10 and bound the S100A10 3′UTR, increasing mRNA stability and 3′UTR‐dependent reporter activity. RBM4 overexpression promoted migration/invasion and reduced apoptosis, whereas S100A10 knockdown reversed these phenotypes. In the TIME context, RBM4 reduced CD8 + T‐cell effector function (lower Granzyme B/IFN‐γ/TNF‐α) while elevating IL‐10/TGF‐β, accompanied by diminished CD8 + infiltration and fewer CD8 + GZMB + /CD8 + TNF‐α + cells in vivo. Mechanistically, RBM4 activated the cPLA2/5‐LOX axis (increased phospho‐forms with stable totals). S100A10 silencing or PD‐1 blockade mitigated immune suppression and restrained tumor growth, indicating pathway specificity and therapeutic tractability. RBM4 enhances S100A10 mRNA stability, remodels the HCC TIME, suppresses CD8 + T‐cell effector function, and promotes immune evasion; targeting the RBM4–S100A10 axis may improve responses to PD‐1 immunotherapy.
ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is largely refractory to immune checkpoint blockade. Conventional Mendelian randomization (MR) using steady‐state expression quantitative trait loci (eQTLs) may miss regulatory effects that emerge during CD4 + T‐cell activation. We used a two‐stage genetic framework. Fixed‐effect meta‐analysis across four PDAC GWAS served as an exploratory cross‐cohort discovery screen, followed by GCST90041814 as the ancestry‐matched European primary analysis. Associations were assessed by colocalization, summary‐data‐based MR with HEIDI testing, and DICE/eQTLGen replication. Paired sample‐level pseudobulk analysis evaluated tumor‐adjacent CD4 + T cells. Bulk transcriptomic, mediation, virtual perturbation, survival and phenome‐wide analyses were treated as exploratory. The pooled discovery screen identified 67 genes at FDR < 0.05; 65 also showed concordant effect directions across all four cohorts and constituted the main discovery set. In the European primary analysis, 66 of 1247 genes had nominal p < 0.05, but none survived cohort‐wide FDR correction. SMDT1 (European OR = 0.603, 95% CI 0.412–0.884, p = 0.009, FDR = 0.754) and CMTM7 (European OR = 1.208, 95% CI 1.026–1.424, p = 0.023, FDR = 0.754) retained directionally consistent nominal associations and were prioritized as exploratory candidates. In six paired GSE212966 samples, pseudobulk changes were directionally consistent but not significant (SMDT1 log2FC = −0.381, p = 0.250; CMTM7 log2FC = 0.493, p = 0.199). Dynamic CD4 + T‐cell eQTL MR nominates SMDT1 and CMTM7 as candidate genes associated with PDAC susceptibility. The pooled discovery evidence and directionally consistent nominal European associations support prioritization, but no gene survived FDR correction in the European analysis. Transcriptomic and downstream functional findings remain exploratory and require experimental validation.
