Bao Jing, Sun Rui, Pan Zhenjiang, Wei Shepeng
Department of Neurosurgery, Shidong Hospital, Yangpu District, Shidong Hospital Affiliated to University of Shanghai for Science and Technology, No. 999, Shiguang Road, Yangpu District, Shanghai, 200438, China.
Naunyn Schmiedebergs Arch Pharmacol. 2025 Jan;398(1):919-931. doi: 10.1007/s00210-024-03327-w. Epub 2024 Aug 1.
To explore how the ubiquitin-conjugating enzyme E2D3 (UBE2D3) influences temozolomide (TMZ) resistance in glioblastoma (GBM), and to clarify the association between UBE2D3 and WTAP. The UBE2D3 protein expression in GBM tissues were detected using immunohistochemistry (IHC) through tissue microarrays. The potential pathways of UBE2D3 in TCGA-GBM were predicted via Gene Set Enrichment Analysis (GSEA). To investigate UBE2D3's role in TMZ resistance, GBM cells were transduced with UBE2D3 shRNA or overexpression lentivirus, followed by assessments of CCK-8, flow cytometry, comet assay, and western blot analysis. Furthermore, a subcutaneous tumor model was established in nude mice using U87 cells transduced with interfering lentivirus to observe tumor growth and assess cell apoptosis using TUNEL staining. Mechanically, mA content analysis, mA methylated RNA immunoprecipitation quantitative PCR, reporter gene assay, mRNA stability measurements, RNA immunoprecipitation, quantitative Real-Time PCR, and Western blot assays were carried out to verify the role of WTAP/IGF2BP1 in regulating UBE2D3 expression. UBE2D3 exhibited elevated expression levels in GBM tissues compared with normal brain tissues and was associated with the DNA repair signaling pathway. In both in vitro and in vivo studies, it was demonstrated that TMZ treatment combined with reduced UBE2D3 expression further suppressed U87 cell viability and tumor growth, with a notable increase in apoptosis rate and DNA damage. Conversely, the overexpression of UBE2D3 had the opposite impact. Furthermore, our findings revealed that WTAP promotes the mA modification of UBE2D3 via an IGF2BP1-dependent mechanism. The WTAP-IGF2BP1 axis regulates UBE2D3 stability in an mA-dependent manner, influencing tumor malignancy and TMZ chemosensitivity in GBM via the DNA repair signaling pathway.
探讨泛素结合酶E2D3（UBE2D3）如何影响胶质母细胞瘤（GBM）对替莫唑胺（TMZ）的耐药性，并阐明UBE2D3与WTAP之间的关联。通过组织芯片免疫组化（IHC）检测GBM组织中UBE2D3蛋白表达。通过基因集富集分析（GSEA）预测TCGA-GBM中UBE2D3的潜在通路。为研究UBE2D3在TMZ耐药中的作用，用UBE2D3 shRNA或过表达慢病毒转导GBM细胞，随后进行CCK-8、流式细胞术、彗星试验和蛋白质免疫印迹分析。此外，使用转导干扰慢病毒的U87细胞在裸鼠中建立皮下肿瘤模型，观察肿瘤生长并使用TUNEL染色评估细胞凋亡。机制上，进行了mA含量分析、mA甲基化RNA免疫沉淀定量PCR、报告基因测定、mRNA稳定性测量、RNA免疫沉淀、定量实时PCR和蛋白质免疫印迹分析，以验证WTAP/IGF2BP1在调节UBE2D3表达中的作用。与正常脑组织相比，UBE2D3在GBM组织中表达水平升高，且与DNA修复信号通路相关。在体外和体内研究中均表明，TMZ治疗联合降低UBE2D3表达进一步抑制U87细胞活力和肿瘤生长，凋亡率和DNA损伤显著增加。相反，UBE2D3过表达则产生相反的影响。此外，我们的研究结果表明，WTAP通过依赖IGF2BP1的机制促进UBE2D3的mA修饰。WTAP-IGF2BP1轴以mA依赖的方式调节UBE2D3稳定性，通过DNA修复信号通路影响GBM中的肿瘤恶性程度和TMZ化学敏感性。
