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Updated: Mar 30, 2026
A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
miR-17 is involved in Japanese Flounder (Paralichthys olivaceus) development by targeting the Cdc42 mRNA
Hongmei Zhang1, Yuanshuai Fu1, Zhiyi Shi1
1Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China.
Abstract:
The expression patterns of 197 miRNAs during Japanese flounder metamorphic development were recently analyzed. miR-17 was differentially expressed during the metamorphic period of the Japanese flounder; however, the role of miR-17 in Japanese flounder development has remained elusive to date. Bioinformatics analysis showed that Cdc42 was a putative target of miR-17. Cdc42 is a gene related to cell adhesion, migration, polarity, cytokinesis, growth, actin cytoskeleton, microtubule dynamics and transcription factor activity; thus, Cdc42 may contribute to metamorphic development. In our study, overexpression of miR-17 in FEC cells suppressed Cdc42 expression. The luciferase reporter assay confirmed that Cdc42 was the target of miR-17. The Cdc42 cDNA from the Japanese flounder was cloned and characterized for the first time. The expression of miR-17 was found to be negatively correlated with Cdc42 mRNA expression during temporal development and in the tissues of adult Japanese flounders. These results indicated that the decrease in miR-17 contributed to the up-regulation of Cdc42 during Japanese flounder metamorphosis. Cdc42 gene expression was down-regulated by thyroid hormone during Japanese flounder metamorphosis, whereas miR-17 was significantly up-regulated by thyroid hormone during these stages. These results indicated that miR-17 was a negative regulator of Cdc42.
Insights
MicroRNA-17 (miR-17) regulates Cdc42 expression during Japanese flounder metamorphosis. This study reveals miR-17 as a negative regulator, impacting flounder development by controlling Cdc42 levels.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) play crucial roles in regulating gene expression during animal development.
- Japanese flounder (Paralichthys olivaceus) metamorphosis is a complex process involving significant physiological and morphological changes.
- The specific roles of many miRNAs, including miR-17, in fish metamorphosis remain largely uncharacterized.
Purpose of the Study:
- To investigate the role of miR-17 in Japanese flounder development.
- To identify and validate the target gene(s) of miR-17 during metamorphosis.
- To elucidate the regulatory relationship between miR-17, Cdc42, and thyroid hormone during Japanese flounder metamorphosis.
Main Methods:
- Bioinformatics analysis to predict miRNA targets.
- Overexpression of miR-17 in fish embryonic cell (FEC) lines.
- Luciferase reporter assays to confirm miRNA-target interaction.
- Quantitative real-time PCR (qRT-PCR) for gene expression analysis.
- Cloning and characterization of Japanese flounder Cdc42 cDNA.
Main Results:
- Bioinformatics predicted Cdc42 as a target of miR-17.
- Overexpression of miR-17 suppressed Cdc42 expression in FEC cells.
- Luciferase assays confirmed Cdc42 as a direct target of miR-17.
- miR-17 expression was negatively correlated with Cdc42 mRNA levels during flounder development and in adult tissues.
- Thyroid hormone up-regulated miR-17 while down-regulating Cdc42 during metamorphosis.
Conclusions:
- miR-17 directly targets and negatively regulates Cdc42 expression in Japanese flounder.
- The miR-17/Cdc42 regulatory axis is involved in Japanese flounder metamorphic development.
- Thyroid hormone mediates the expression of both miR-17 and Cdc42, suggesting a coordinated regulatory mechanism during metamorphosis.
