Department of Pediatrics, Second Hospital Cheeloo College of Medicine, Shandong University, No. 247 Beiyuan Street, Jinan, People's Republic of China.
Department of Pediatric Internal Medicine, The Second Affiliated Hospital of Shandong First Medical University, Taian, People's Republic of China.
Mol Cell Biochem. 2021 Jun;476(6):2409-2420. doi: 10.1007/s11010-021-04090-9. Epub 2021 Feb 18.
MiR-200a acts as a key role in tumor malignant progression. This work purposed to assess the function of miR-200a in Wilm's tumor. Based on bioinformatics analysis, the expression, prognostic value and related pathways of miR-200a and CDC7 (a potential downstream molecule of miR-200a) in Wilm's tumor were analyzed. qRT-PCR was conducted to confirm the miR-200a level in Wilm's tumor cells. The luciferase reporter assay was carried out to verify the binding of miR-200a to 3'-UTR of CDC7. Then, the impacts of miR-200a and CDC7 on cell viability and apoptosis were measured using CCK-8 and flow cytometry assays. Also, western blot was applied to measure the expression of CDC7 as well as Wnt/β-catenin signaling pathway-related proteins and apoptosis proteins. Herein, we revealed that miR-200a was lowly expressed in Wilm's tumor tissues and cells and the low miR-200a expression is closely bound up with death and poor outcomes. Moreover, miR-200a directly targeted and inhibited CDC7 in Wilm's tumor cells. Biological function experiments illustrated that overexpression of miR-200a reduced the viability and elevated the apoptosis of Wilm's tumor cells, while overexpression of CDC7 reversed the inhibitory impact of miR-200a on cell viability and the promoting impact of miR-200a on cell apoptosis. Besides, we revealed that miR-200a/CDC7 axis can decrease the expression of β-Catenin, Cyclin D1 and C-Myc as well as the phosphorylation of GSK-3β, thus inhibiting the Wnt/β-catenin signaling pathway. Furthermore, blocking the Wnt/β-catenin signaling pathway caused an increase on cell apoptosis, while overexpression of CDC7 can reverse these impacts. Collectively, miR-200a/CDC7 axis involved in regulating the malignant phenotype of Wilm's tumor through Wnt/β-catenin signaling pathway, which provides a theoretical basis for targeted molecular therapy of Wilm's tumor.
miR-200a 在肿瘤恶性进展中起关键作用。本研究旨在评估 miR-200a 在肾母细胞瘤中的功能。通过生物信息学分析，分析了 miR-200a 和 CDC7（miR-200a 的潜在下游分子）在肾母细胞瘤中的表达、预后价值和相关途径。通过 qRT-PCR 检测肾母细胞瘤细胞中 miR-200a 的水平。通过荧光素酶报告实验验证 miR-200a 与 CDC7 3'-UTR 的结合。然后，通过 CCK-8 和流式细胞术检测 miR-200a 和 CDC7 对细胞活力和凋亡的影响。此外，通过 Western blot 检测 CDC7 以及 Wnt/β-catenin 信号通路相关蛋白和凋亡蛋白的表达。结果显示，miR-200a 在肾母细胞瘤组织和细胞中低表达，低表达的 miR-200a 与死亡和不良预后密切相关。此外，miR-200a 可直接靶向并抑制肾母细胞瘤细胞中的 CDC7。生物学功能实验表明，miR-200a 的过表达降低了肾母细胞瘤细胞的活力，促进了细胞凋亡，而过表达的 CDC7 则逆转了 miR-200a 对细胞活力的抑制作用和对细胞凋亡的促进作用。此外，我们发现 miR-200a/CDC7 轴可降低β-Catenin、Cyclin D1 和 C-Myc 的表达以及 GSK-3β 的磷酸化，从而抑制 Wnt/β-catenin 信号通路。此外，阻断 Wnt/β-catenin 信号通路会导致细胞凋亡增加，而过表达的 CDC7 可以逆转这些影响。综上所述，miR-200a/CDC7 轴通过 Wnt/β-catenin 信号通路参与调节肾母细胞瘤的恶性表型，为肾母细胞瘤的靶向分子治疗提供了理论基础。
