DNMT3A Regulates miR-149 DNA Methylation to Activate NOTCH1/Hedgehog Pathway to Promote the Development of Junctional Osteosarcoma

Abstract

Purpose. To investigate the DNMT3A/miR-149/NOTCH1/Hedgehog axis regulating the development of osteosarcoma. Methods. 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. Results. 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. Conclusion. 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.

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“…Among these, DNA methyltransferases (DNMTs), particularly DNMT3A, have been pinpointed as critical in suppressing miR-149 expression via DNA methylation. This suppression subsequently activates the NOTCH1 and Hedgehog pathways, fueling osteosarcoma growth and metastasis [ 30 ]. The emerging role of long non-coding RNAs (lncRNAs) in this context is evident in molecules like THAP9-AS1.…”

CircDOCK1 Regulates miR-186/DNMT3A to Promote Osteosarcoma Progression

“…The enhanced catalytic activity of DNMT3A is associated with cancer development and progression. Cheng et al reported that DNMT3A facilitated OS progression by mediating miR-149 DNA methylation to activate the Notch1/Hedgehog pathway [ 43 ]. In line with the former study, DNMT3A’s carcinogenic role in OS was also recognized herein, and miR-186 suppressed OS by decreasing DNMT3A.…”

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“…Further analysis of OS cells showed hypermethylation of CGI CpG79 upstream of miR-486-5p [ 17 ]. In a study by Cheng and Wang et al, 2022, DNA methylation was similarly found to be responsible for the suppression of miRNA [ 18 ]. miR-149 was underexpressed in OS samples, and less expression correlated with higher-stage tumors.…”