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.

Figure 1

miR-149 was significantly low expressed in osteosarcoma tissues and cells. (a) Differentially expressed microRNA in microRNA expression microarray GSE67268 of osteosarcoma tissues. (b) qRT-PCR detection of miR-149 expression levels in cancer tissues and paired paraneoplastic tissues of 42 osteosarcoma patients. (c–e) Analysis of miR-149 451a expression levels in relation to clinical stage, lymph node metastasis, and differentiation level of osteosarcoma patients. (f) miR-149 expression levels in normal human esophageal epithelial cells HET-1A and osteosarcoma cell lines SAOS-2, osteosarcoma 9706, and MG63. In (b–e), each point represents one sample, and data were analyzed for differences using the paired or unpaired test; ^∗∗P < 0.01, ^∗∗∗P < 0.001. In (f), data were analyzed for differences using one-way ANOVA and Tukey's multiple comparison test for analysis of variance on data; ^∗∗∗P < 0.001.

Figure 2

Exogenous overexpression of miR-149 significantly inhibits the growth and metastasis of osteosarcoma cells in vitro. (a) qRT-PCR to detect the expression level of miR-149 in MG63 and SAOS-2 cells after transfection with mimic control or miR-149 mimic. (b) Plate cloning assay to detect the number of clones formed in MG63 and SAOS-2 cells. The number of clones formed. (c) Caspase-3 kit to detect changes in caspase-3 activity in MG63 and SAOS-2 cells. (d) Flow cytometry to detect the proportion of apoptosis in MG63 and SAOS-2 cells. (e) qRT-PCR to detect the EMT-related factors E-cadherin, ZO-1, vimentin, and N-cadherin mRNA expression levels. (f, g) Transwell assay to detect the migration and invasion ability of MG63 and SAOS-2 cells. Each experiment was repeated three times, and data were presented as mean plus or minus standard deviation, and in (a–g), 2-way ANOVA and Tukey's multiple comparison test were used to analyze the data for differences, ^∗∗∗P < 0.001.

Figure 3

miR-149 promoter has significant methylation modification. (a) MSP-qPCR detects the methylation level of miR-149 promoter in cancerous and paraneoplastic tissues of 42 osteosarcoma patients. (b) Correlation between miR-149 and its DNA methylation level in tumor tissues of osteosarcoma patients. (c) MSP-qPCR detects the correlation between miR-149 and its DNA methylation level in normal tissues of osteosarcoma patients. Methylation levels of the promoter of miR-149 in tumor tissues from osteosarcoma patients. (d) Correlation between miR-149 and its DNA methylation levels in tumor tissues from osteosarcoma patients. Each point in CD represents one sample, and the data were tested for significant differences using the paired t-test, ^∗∗P < 0.01. In (e, f), each experiment was repeated three times, and the data were presented as mean plus or minus standard deviation, using one-way or 2-way ANOVA and Tukey's multiple comparison test for analysis of variance on data, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 4

DNMT3A promotes miR-149 DNA methylation modification. (a) Pearson correlation test to analyze the correlation analysis between miR-149 and DNMT3A expression levels. (b) HPA website to retrieve the staining intensity of DNMT3A in normal esophageal tissues and osteosarcoma tissue samples by immunohistochemistry. (c) ChIP-qPCR to detect DNMT3A binding relationship with miR-149 promoter. (d) The luciferase reporter vector pGL3-enhancer containing miR-149 promoter was constructed, cotransfected into 293T cells with different doses of DNMT3A overexpression plasmids, and the expression level of miR-149 in the cells was detected. In (a), each point represents one sample, and the data were tested for significant differences using paired t-test, ^∗∗P < 0.01. In (e, f), each experiment was repeated three times, and the data were presented as mean plus minus standard deviation using one-way or 2-way ANOVA and Tukey's multiple comparison test for analysis of variance on data; ^∗∗P < 0.01, ^∗∗∗P < 0.001.