Stem Cell Research & Therapy•2025•DOI: 10.1186/s13287-025-04151-6
Background Autosomal dominant osteodystrophy type II (ADO2) is an inherited disease characterized by an abnormal increase in bone mineral density, and CLCN7 (R286W) is its most common causative mutation. The aim of this study was to explore the new idea of siRNA technology applied to the in vitro treatment of ADO2. Methods Urinary-derived cells from ADO2 patients were collected to establish induced pluripotent stem cells (iPSCs) model. The siRNA targeting CLCN7 (R286W) mutant mRNA was designed. the cytotoxicity of the delivery vector DMPC-SPIONs was comprehensively evaluated by CCK-8 assay, flow cytometry and scratch assay. Finally, qPCR was utilized to verify the post-transcriptional silencing effect of siRNAs. Results We found that DMPC-SPIONs had low cytotoxicity and were able to effectively deliver siRNAs into ADO2-iPSCs. qPCR confirmed that siRNA-DMPC-SPIONs were able to significantly reduce the expression level of mutant CLCN7 (66%), while there was no significant effect on the expression of wild-type CLCN7. Conclusions This study developed a gene silencing strategy based on siRNAs and DMPC-SPIONs, which provides a potential new approach for the treatment of ADO2 and demonstrates the potential application of siRNA technology in the treatment of autosomal dominant genetic diseases. Innovative statements In this study, we used the established ADO2-iPSCs using patient's urine-derived cells to explore the safety and efficacy of siRNA technology based on the principle of RNA interference for ADO2 treatment for the first time. In addition, we chose DMPC-SPIONs as the delivery vehicle for siRNA, which cleverly exploits the advantages of nanoparticles such as superparamagnetism, low cytotoxicity, and good bio-histocompatibility.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025019
Cancer cells evade immune detection through checkpoint molecules like PD-L1 and PD-L2 which suppress T-cell activation. While PD-L1 is well-studied, the role of PD-L2 remains unclear. Pyruvate kinase M2 (PKM2), a metabolic enzyme, influences immune checkpoint regulation, but its role in PD-L1 and PD-L2 modulation is not well defined. Here, we investigate the role of pyruvate kinase M2 (PKM2) in modulating the immune checkpoint molecules PD-L1 and PD-L2 via GATA3 in cancer cells, with insights from both human and mouse models. We find that PKM2 enhances PD-L1 expression while inhibiting PD-L2, a dual regulatory mechanism that facilitates immune evasion. Knockdown and overexpression experiments revealed GATA3 as a key mediator. PKM2 knockout reduced GATA3 level, leading to decreased PD-L1 and increased PD-L2 expression. Chromatin immunoprecipitation (ChIP)-qPCR demonstrates that GATA3 functions as a direct transcription factor capable of binding to the promoters of PD-L1 and PD-L2. In silico analyses of 81 esophageal squamous cell carcinoma (ESCC) cases from the TCGA database demonstrate that PKM2 mRNA is unrelated to PD-L1 and PD-L2 expression but is negatively correlated with CD8+ T-cell infiltration in ESCC. To further validate these findings, we establish a xenograft model using immune-competent C57/BL6N mice, where knockdown of PKM2 results in significant downregulation of both PD-L1 and PD-L2 expression. Collectively, these findings underscore the divergent roles of PKM2 in regulating immune checkpoint expression in human and mouse cancer models and suggest that targeting the PKM2-GATA3 axis could enhance cancer immunotherapy by fine-tuning PD-L1 and PD-L2 levels.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025019
Cancer cells evade immune detection through checkpoint molecules PD-L1 and PD-L2, which suppress T-cell activation. While PD-L1 is well-studied, the role of PD-L2 remains unclear. Pyruvate kinase M2 (PKM2), a metabolic enzyme, influences immune checkpoint regulation, but its role in PD-L1 and PD-L2 modulation is not well defined. Here, we investigate the role of PKM2 in modulating PD-L1 and PD-L2 via GATA3 in cancer cells, with insights from both human and mouse models. We find that PKM2 enhances PD-L1 expression while inhibiting PD-L2, a dual regulatory mechanism that facilitates immune evasion. Knockdown and overexpression experiments revealed GATA3 as a key mediator. PKM2 knockout reduced GATA3 level, leading to decreased PD-L1 and increased PD-L2 expression. Chromatin immunoprecipitation (ChIP)-qPCR demonstrates that GATA3 functions as a direct transcription factor capable of binding to the promoters of PD-L1 and PD-L2. In silico analyses of 81 esophageal squamous cell carcinoma (ESCC) cases from the TCGA database demonstrate that PKM2 mRNA is unrelated to PD-L1 and PD-L2 expression but is negatively correlated with CD8+ T-cell infiltration in ESCC. To further validate these findings, we establish a xenograft model using immune-competent C57/BL6N mice, where knockdown of PKM2 results in significant downregulation of both PD-L1 and PD-L2 expression. Collectively, these findings underscore the divergent roles of PKM2 in regulating immune checkpoint expression in human and mouse cancer models and suggest that targeting the PKM2-GATA3 axis could enhance cancer immunotherapy by fine-tuning PD-L1 and PD-L2 levels.