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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
MicroRNA-542-3p suppresses tumor cell proliferation via targeting Smad2 inhuman osteosarcoma
Yinsheng Wu1, Jiongming You1, Feng Li1
1Department of Orthopedic Surgery, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou, Zhejiang 325000, P.R. China.
Abstract:
Osteosarcoma (OS) is an aggressive malignant neoplasm that arises from primitive transformed cells of mesenchymal origin, and that exhibits osteoblastic differentiation and produces malignant osteoid. MicroRNA (miR)-542-3p has been reported to serve a crucial role in the initiation and development of several types of human cancer. However, whether miR-542-3p is involved in tumorigenesis and tumor progression of OS remains unclear. In the present study, the expression of miR-542-3p in OS cells and patients with OS, and its functional mechanism in OS were investigated. The data demonstrated that expression of miR-542-3p was significantly decreased in OS tissues and cell lines, and that restoration of miR-542-3p expression in OS cells inhibited cell proliferation and induced cell apoptosis. Through bioinformatics analysis and luciferase reporter assays, the present study identified that miR-542-3p directly targeted Smad2 mRNA and negatively regulated the expression of Smad2 at the protein level in OS cells. Furthermore, it was confirmed that OS tumor tissues with a low expression of miR-542-3p exhibited markedly higher Smad2 expression. Finally, through the use of gain of function and rescue experiments, the present study demonstrated that restoration of miR-542-3p was able to suppress the growth and proliferation of OS cells through directly targeting Smad2. To the best of our knowledge, this is the first study to demonstrate that decreased expression of miR-542-3p serves a role in tumor suppression in OS pathogenesis through targeting Smad2. These results will aid in elucidating the functions of miR-542-3p, and suggest that miR-542-3p may serve as a tumor suppressor gene and a promising therapeutic target of OS.
Insights
MicroRNA (miR)-542-3p is downregulated in osteosarcoma (OS). Restoring miR-542-3p suppresses OS cell growth by targeting Smad2, indicating its potential as a tumor suppressor and therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Osteosarcoma (OS) is an aggressive bone cancer.
- MicroRNA (miR)-542-3p's role in OS is not well understood.
- Investigating miR-542-3p's function in OS tumorigenesis is crucial.
Purpose of the Study:
- To investigate the expression and mechanism of miR-542-3p in osteosarcoma.
- To determine if miR-542-3p acts as a tumor suppressor in OS.
- To identify potential therapeutic targets for OS.
Main Methods:
- Quantitative real-time PCR to measure miR-542-3p expression.
- Cell proliferation and apoptosis assays.
- Bioinformatics analysis, luciferase reporter assays, and Western blotting to identify miR-542-3p targets.
- Gain-of-function and rescue experiments.
Main Results:
- miR-542-3p expression is significantly decreased in OS tissues and cell lines.
- Restoring miR-542-3p inhibits OS cell proliferation and induces apoptosis.
- miR-542-3p directly targets Smad2 mRNA, reducing Smad2 protein levels.
- Low miR-542-3p expression correlates with high Smad2 expression in OS tissues.
Conclusions:
- Decreased miR-542-3p expression promotes osteosarcoma progression by upregulating Smad2.
- miR-542-3p functions as a tumor suppressor in OS.
- miR-542-3p is a potential therapeutic target for osteosarcoma treatment.
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