Department of Ophthalmology, Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China.
The Second Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China.
Drug Dev Res. 2024 Aug;85(5):e22222. doi: 10.1002/ddr.22222.
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.
视网膜母细胞瘤（RB）是一种儿科恶性肿瘤，通常在出生时或幼儿期诊断。RB 的发病机制以基本螺旋-环-螺旋（BHLH）转录因子 MYCN 的扩增为特征，MYCN 作为一种转录调节剂，能够与 Dickkopf 3（DKK3）结合。然而，MYCN 引起的 RB 细胞恶性进展中 DKK3 的精确作用仍然难以捉摸。在本研究中，在 RB 细胞中过表达或干扰 MYCN 的表达。随后，通过定量实时聚合酶链反应和 Western blot 分析评估 DKK3 的表达水平。通过细胞计数试剂盒-8 测定和 5-乙炔基-2'-脱氧尿苷染色评估细胞增殖，通过流式细胞术和 Western blot 分析分别分析细胞周期进程和细胞凋亡。此外，通过 Western blot 分析评估参与 Wnt/β-catenin/Fra-1/p53 信号通路的蛋白质的表达。为了获得进一步的见解，引入了 Wnt 激动剂和 P53 抑制剂 PFT-α 进行探索。目前的研究表明，在 RB 细胞中，MYCN 的表达水平与 DKK3 的表达水平呈负相关。此外，在高表达 MYCN 的 RB 细胞中，DKK3 的过表达抑制细胞增殖，促进细胞凋亡，并使细胞周期停滞。此外，通过调节 Wnt/βcatenin/Fra-1/p53 信号通路，增强 DKK3 的表达抑制 RB 细胞的增殖，促进细胞周期停滞和细胞凋亡。此外，体内实验表明 DKK3 的过表达抑制 RB 肿瘤的生长。总之，我们的研究结果表明，MYCN 通过抑制 DKK3 的表达来刺激 Wnt/β-catenin/Fra-1 通路，从而抑制 p53 的活性，促进 RB 的恶性进展。
