Abstract：
Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed through quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using the Cell Counting Kit-8 assay and 5-ethynyl-2\'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/β-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/βcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/β-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.
摘要：
视网膜母细胞瘤（RB）是一种儿科恶性肿瘤，通常在出生时或儿童早期被诊断。RB的发病机制以碱性螺旋-环-螺旋（BHLH）转录因子MYCN的扩增为标志，其充当能够与Dickkopf3(DKK3)结合的转录调节因子。然而,DKK3在由MYCN引起的RB细胞恶性进展中的确切作用仍然难以捉摸。在本研究中,MYCN的表达在RB细胞中被过表达或被干扰。随后，通过实时定量聚合酶链反应和蛋白质印迹分析评估DKK3的表达水平.使用细胞计数试剂盒-8测定和5-乙炔基-2'-脱氧尿苷染色评估细胞增殖,而细胞周期进程和细胞凋亡通过流式细胞术和蛋白质印迹分析，分别。此外,通过Westernblot分析评估Wnt/β-catenin/Fra-1/p53信号通路相关蛋白的表达.为了获得更多的见解，将Wnt激动剂和P53抑制剂PFT-α引入到探索中。目前的研究表明RB细胞中MYCN和DKK3的表达水平之间呈负相关。此外,DKK3过表达抑制细胞增殖,促进细胞凋亡，并在高表达MYCN的RB细胞中阻滞细胞周期。此外，DKK3表达增强抑制增殖，通过调节wnt/βcatenin/Fra-1/p53信号通路促进RB细胞的细胞周期阻滞和凋亡。此外，体内实验表明，DKK3的过表达抑制了RB肿瘤的生长。总的来说，我们的发现阐明了MYCN通过抑制DKK3表达刺激Wnt/β-catenin/Fra-1通路,最终抑制p53活性并促进RB的恶性进展。
