The raw images of western blot.

PurposeWilms’ tumor (WT), also known as nephroblastoma, is the predominant form of primary malignant renal cancer. The unfavorable prognoses linked to anaplastic nephroblastoma and recurrent nephroblastoma emphasize the crucial requirement for the exploration of innovative treatment modalities for WT.MethodsOur study conducted one-way Cox regression and Kaplan-Meier analyses using TARGET-WT nephroblastoma data to identify differentially expressed genes in nephroblastoma and evaluate their prognostic relevance. Utilizing the Connectivity Map database, ZSTK474 emerged as a viable therapeutic option for WT. The effect of ZSTK474 on WT and related underlying mechanisms were further investigated through in vitro and in vivo investigations.ResultsThe in vivo experiment results indicated that ZSTK474 effectively inhibited subcutaneous tumor growth in WT mice. CCK-8 assays revealed two nephroblastoma cell lines exhibited half-inhibitory concentrations of 2μM and 2.51μM for ZSTK474, respectively. ZSTK474 was shown to inhibit the migration and invasion capabilities of WT cells in both Transwell and wound healing assays. Flow cytometry apoptosis and TUNEL assays demonstrated that ZSTK474 induced apoptosis in WT cells. Cell cycle analysis revealed that ZSTK474 led to the induction of G0/G1 phase arrest. Sequencing of ZSTK474-treated WiT49 cells suggested that the impact of ZSTK474 on WT might be mediated by the PI3K/Akt pathway, specifically by inhibiting PIK3R3. Knock-down of PIK3R3 confirmed that ZSTK474 downregulated PIK3R3, reducing Akt phosphorylation, cyclin D and CDK4 levels and elevating P21 expression in nephroblastoma cells. However, current research has limitations, including a lack of understanding of the long-term effects and potential resistance mechanisms of new therapies.ConclusionThis research provides insight into the potential of ZSTK474 and other PI3K inhibitors for treating nephroblastoma.

研究背景：威尔姆斯瘤（Wilms’ tumor, WT）又称肾母细胞瘤，是原发性肾脏恶性肿瘤的主要类型。间变性肾母细胞瘤与复发性肾母细胞瘤相关的不良预后，凸显了探索WT创新治疗手段的迫切需求。
研究方法：本研究利用TARGET-WT肾母细胞瘤数据集开展单因素Cox回归与Kaplan-Meier分析，以筛选肾母细胞瘤差异表达基因并评估其预后相关性。通过连通性图谱（Connectivity Map）数据库筛选，ZSTK474被确定为WT潜在治疗药物。随后通过体外与体内实验，进一步探究ZSTK474对WT的作用效果及潜在分子机制。
研究结果：体内实验结果显示，ZSTK474可有效抑制WT小鼠皮下肿瘤生长。CCK-8实验表明，两种肾母细胞瘤细胞系对ZSTK474的半抑制浓度分别为2μM与2.51μM。Transwell实验与划痕愈合实验均证实，ZSTK474可抑制WT细胞的迁移与侵袭能力。流式细胞术凋亡检测与TUNEL实验结果显示，ZSTK474可诱导WT细胞凋亡。细胞周期分析结果表明，ZSTK474可使WT细胞阻滞于G0/G1期。对ZSTK474处理后的WiT49细胞进行测序分析，提示ZSTK474对WT的作用可能通过PI3K/Akt通路介导，具体机制为抑制PIK3R3。敲低PIK3R3实验证实，ZSTK474可下调PIK3R3的表达，降低肾母细胞瘤细胞中Akt磷酸化水平、细胞周期蛋白D与CDK4的表达量，并升高P21的表达水平。然而，本研究尚存局限性，例如尚未明确新疗法的长期效应与潜在耐药机制。
研究结论：本研究揭示了ZSTK474及其他PI3K抑制剂用于治疗肾母细胞瘤的潜在可能性。