Department of Orthopedics, Loudi Central Hospital of Hunan Province, Loudi, Hunan, 417000, China.
Department of Orthopedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, 410011, China.
Biomed Res Int. 2022 Jul 21;2022:3261213. doi: 10.1155/2022/3261213. eCollection 2022.
To investigate the DNMT3A/miR-149/NOTCH1/Hedgehog axis regulating the development of osteosarcoma.
First, microRNA and mRNA expression microarrays were downloaded from the GEO database for osteosarcoma and differentially expressed microRNAs were analyzed. Subsequently, we collected cancerous tissues and corresponding paracancerous tissues from 42 osteosarcoma patients and examined the expression levels of miR-149, DNMT3A, and NOTCH1 in the samples. Subsequently, miR-149 was overexpressed in osteosarcoma cells to detect cell proliferation and metastatic ability changes. We then queried the methylation level of the miR-149 promoter on the bioinformatics website and verified it by experiment. We further demonstrated the expression level of miR-149 with NOTCH1 using a dual luciferase assay and confirmed the role of NOTCH1 on osteosarcoma cell growth and metastasis by functional rescue assay. Finally, we detected the activation level of the Hedgehog/catenin signaling pathway by WB and immunofluorescence.
miR-149 was significantly low expressed in osteosarcoma tissues and cells, while DNMT3A and NOTCH1 were highly expressed in osteosarcoma tissues and cells, and negatively correlated with miR-149 expression levels. Overexpression of miR-149 significantly inhibited the growth and metastasis of osteosarcoma cells in vitro and in vivo, and we found that DNMT3A could promote the methylation modification of the miR-149 promoter, thereby inhibiting the expression of miR-149. Subsequently, the experimental results showed that miR-149 could target negative regulation of NOTCH1, and further overexpression of NOTCH1 in cells with high miR-149 expression could promote the growth and metastasis of osteosarcoma cells in vitro.
The methyltransferase DNMT3A suppresses miR-149 expression by promoting methylation modification of the miR-149 promoter, resulting in elevated expression levels of NOTCH1 in cells, therefore exacerbating activation of the Hedgehog signaling pathway and therefore exacerbating the development and progression of osteosarcoma.
研究 DNMT3A/miR-149/NOTCH1/Hedgehog 轴在骨肉瘤发生发展中的调控作用。
首先，从 GEO 数据库下载骨肉瘤的 microRNA 和 mRNA 表达微阵列，分析差异表达的 microRNA。随后，我们收集了 42 例骨肉瘤患者的癌组织及相应癌旁组织，检测样本中 miR-149、DNMT3A 和 NOTCH1 的表达水平。随后，在骨肉瘤细胞中过表达 miR-149，检测细胞增殖和转移能力的变化。我们在生物信息学网站上查询了 miR-149 启动子的甲基化水平，并通过实验进行了验证。我们进一步通过双荧光素酶报告实验证实了 miR-149 与 NOTCH1 的表达水平，并用功能拯救实验证实了 NOTCH1 对骨肉瘤细胞生长和转移的作用。最后，我们通过 WB 和免疫荧光检测 Hedgehog/β-catenin 信号通路的激活水平。
miR-149 在骨肉瘤组织和细胞中表达明显下调，而 DNMT3A 和 NOTCH1 在骨肉瘤组织和细胞中高表达，且与 miR-149 表达水平呈负相关。miR-149 的过表达显著抑制骨肉瘤细胞的体外和体内生长和转移，我们发现 DNMT3A 可以促进 miR-149 启动子的甲基化修饰，从而抑制 miR-149 的表达。随后，实验结果表明，miR-149 可以负向调控 NOTCH1 的表达，并且在 miR-149 高表达的细胞中进一步过表达 NOTCH1 可以促进骨肉瘤细胞的体外生长和转移。
甲基转移酶 DNMT3A 通过促进 miR-149 启动子的甲基化修饰抑制 miR-149 的表达，导致细胞中 NOTCH1 表达水平升高，从而加剧 Hedgehog 信号通路的激活，进而加重骨肉瘤的发生发展。
