Related Experiment Video
Updated: Sep 2, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
DNMT3A Regulates miR-149 DNA Methylation to Activate NOTCH1/Hedgehog Pathway to Promote the Development of Junctional
1Department of Orthopedics, Loudi Central Hospital of Hunan Province, Loudi, Hunan, 417000, China.
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.
Insights
DNA methyltransferase 3A (DNMT3A) suppresses miR-149, increasing NOTCH1 and Hedgehog pathway activation, which drives osteosarcoma development. Restoring miR-149 inhibits osteosarcoma progression.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Osteosarcoma is a primary bone malignancy with complex regulatory mechanisms.
- Understanding the molecular pathways driving osteosarcoma is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the role of the DNMT3A/miR-149/NOTCH1/Hedgehog signaling axis in osteosarcoma development.
- To investigate the regulatory interactions between these key molecules in osteosarcoma progression.
Main Methods:
- Bioinformatic analysis of GEO database for differential microRNA and mRNA expression.
- Expression analysis of miR-149, DNMT3A, and NOTCH1 in patient tissues.
- In vitro functional assays (cell proliferation, metastasis) following miR-149 overexpression.
- Methylation analysis of the miR-149 promoter.
- Dual-luciferase and functional rescue assays to confirm molecular interactions.
- Western blot and immunofluorescence to assess Hedgehog signaling pathway activation.
Main Results:
- miR-149 was significantly downregulated, while DNMT3A and NOTCH1 were upregulated in osteosarcoma.
- DNMT3A promotes miR-149 promoter methylation, suppressing miR-149 expression.
- miR-149 directly targets and downregulates NOTCH1.
- Overexpression of miR-149 inhibited osteosarcoma cell growth and metastasis.
- NOTCH1 overexpression counteracted the inhibitory effects of miR-149, promoting tumor progression.
Conclusions:
- DNMT3A-mediated methylation suppresses miR-149, leading to NOTCH1 upregulation.
- The DNMT3A/miR-149/NOTCH1 axis activates the Hedgehog signaling pathway, promoting osteosarcoma development.
- Targeting this axis holds potential for novel osteosarcoma therapies.
Related Concept Videos
03:14Notch Signaling Pathway
The Notch gene came into the limelight in 1914 after the discovery that its mutation in Drosophila melanogaster leads to a serrated (or "notched") wing margin phenotype. It was not...
01:12Role Of Notch Signalling In Intestinal Stem Cell Renewal
Direct cell-to-cell contact is needed for the activation of Notch signaling. The signal is initiated when a notch ligand binds to a receptor on an adjacent cell, also...
01:37Epigenetic Regulation
X-chromosome...
01:22MicroRNAs
02:23Abnormal Proliferation
03:03mTOR Signaling and Cancer Progression
The mTOR pathway or the...