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MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB

Abstract

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs’ in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3′UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.

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Fig. 1: Based on previous literature (Andersen et al., [27]) followed by bioinformatics analysis, miRmap target score from Fig. S2, miR-432, miR-410, and miR-34a were selected for real-time RT-PCR analysis.
Fig. 2: miR-432-5p down regulates PDGFB expression in OS.
Fig. 3: miR-432-5p targets PDGFB 3′ UTR.
Fig. 4: miR-432-5p/PDGFB signaling regulation in OS regulates endothelial cells proliferation.
Fig. 5: Chick aortic ring sprouting analysis.
Fig. 6: miR-432-5p/PDGFB signaling regulation in OS regulates angiogenesis in sprouting phase.
Fig. 7: miR-432-5p/PDGFB signaling regulation in OS promotes IA in sprouting phase of CAM.
Fig. 8: miR-432-5p/PDGFB signaling regulation in OS regulates angiogenesis in intussusceptive phase.
Fig. 9: miR-432-5p/PDGFB signaling regulation in OS promotes IA in intussusceptive phase of CAM.
Fig. 10: miR-432-5p/PDGFB signaling regulation in OS promotes IA in zebrafish xenograft model.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by Department of Science and Technology, INSPIRE Faculty Program, Government of India for the research grant to SV (grant no. DST/INSPIRE/04/2017/002913).

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SV, RS, SS, and BA performed all the experiments and drafted the manuscript. DA provided technical support for the work. SV secured the funding, designed the work, and analyzed data, and approved the final submitted manuscript.

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Correspondence to Selvaraj Vimalraj.

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Vimalraj, S., Subramanian, R., Saravanan, S. et al. MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB. Lab Invest 101, 1011–1025 (2021). https://doi.org/10.1038/s41374-021-00589-3

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