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Updated: Dec 26, 2025

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
MicroRNA-744 promotes proliferation of osteosarcoma cells by targeting PTEN
Wei Yu1, Peng-Bo Chen2, Fan-Cheng Chen3
1Department of Orthopedic Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China.
Abstract:
MicroRNAs (miRNAs/miRs) are non-coding RNAs that regulate protein synthesis by targeting mRNAs for translational repression or degradation. Previous studies have reported that aberrant expression of miR‑744 may be involved in human osteosarcoma; however, the underlying mechanisms remain elusive. In the present study, the expression levels of miR‑744 and its downstream signals were determined by reverse transcription‑quantitative PCR and western blotting. Cell proliferation was assessed using the bromodeoxyuridine assay, and the target of miR‑744 was investigated using a dual‑luciferase activity assay. The present study identified a significant upregulation of miR‑744 in osteosarcoma tissues compared with adjacent non‑tumor tissues. Furthermore, it was demonstrated that ectopic overexpression of miR‑744 induced by a miR‑744 precursor significantly enhanced proliferation of the osteosarcoma cell line MG63, whereas opposite results were observed following suppression of miR‑744 with its inhibitor. Moreover, as a unique anti‑oncogene, PTEN was identified as a direct target of miR‑744. It was confirmed that miR‑744 downregulated PTEN expression in MG63 cells by targeting the PTEN 3'untranslated region, and that the downstream AKT signal was also regulated by miR‑744. Collectively, the present results suggested that miR‑744 promoted proliferation of human osteosarcoma cells by directly regulating the PTEN/AKT signaling pathway.
Insights
MicroRNAs (miRNAs) promote osteosarcoma cell proliferation by upregulating miR-744. This microRNA targets PTEN, affecting the PTEN/AKT pathway in human bone cancer.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- MicroRNAs (miRNAs) are key regulators of protein synthesis.
- Aberrant miR-744 expression is implicated in human osteosarcoma, but mechanisms are unclear.
Purpose of the Study:
- To investigate the role and mechanism of miR-744 in human osteosarcoma proliferation.
Main Methods:
- Reverse transcription-quantitative PCR and western blotting to measure expression levels.
- Bromodeoxyuridine assay for cell proliferation assessment.
- Dual-luciferase activity assay to identify miR-744 targets.
Main Results:
- miR-744 was significantly upregulated in osteosarcoma tissues.
- miR-744 overexpression enhanced MG63 cell proliferation; inhibition reduced it.
- PTEN was identified as a direct target of miR-744, with miR-744 downregulating PTEN and affecting the AKT pathway.
Conclusions:
- miR-744 promotes human osteosarcoma cell proliferation.
- This promotion occurs via direct regulation of the PTEN/AKT signaling pathway.
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