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Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation
Published on: July 6, 2022
Computational identification and experimental validation of microRNAs binding to the fragile X syndrome gene Fmr1
Xi Gong1, Yanlu Wang, Jianping Zeng
1State Key Laboratory of Food Science and Technology, College of Life Sciences, Nanchang University, Nanchang, 330031, China.
Abstract:
MicroRNAs (miRNAs) usually bind to their target mRNAs through imperfect base pairing in the 3'-untranslated regions (3' UTRs) and regulate target gene expression via post-transcriptional suppression. In recent years, computational approaches to predict miRNA targets have facilitated the identification of potential target sites. In this study, we used three programs TargetScan, miRDB and miRanda to predict potential miRNA binding sites to the fragile X gene Fmr1 and picked out 61 miRNAs which were predicted by all three programs for further investigation. Excitingly, 5 out of these miRNAs, miR-23a, miR-32, miR-124, miR-335-5p and miR-350, were experimentally verified by luciferase reporter assays. Furthermore, overexpression of miR-124 in mouse embryonic neural progenitor cells (eNPC) could not only significantly reduce Fmr1 level, but also increase Cdk4 and cyclin D1 levels which coincidently promoted eNPC proliferation. Our results imply that miR-124 plays an important role in the proliferation of mouse embryonic stem cells by promoting Cdk4 and cyclin D1 expression through directly inhibiting Fmr1 expression.
Insights
MicroRNAs regulate gene expression. This study identified miR-124 as a key regulator of Fmr1, promoting embryonic stem cell proliferation by increasing Cdk4 and cyclin D1 levels.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally.
- Fragile X mental retardation gene (Fmr1) plays a crucial role in neuronal development and function.
- Dysregulation of Fmr1 is implicated in various neurological disorders.
Purpose of the Study:
- To computationally predict and experimentally validate microRNAs targeting the Fmr1 gene.
- To investigate the functional role of a specific microRNA, miR-124, in regulating Fmr1 expression and its impact on neural progenitor cell proliferation.
Main Methods:
- Computational prediction of miRNA binding sites using TargetScan, miRDB, and miRanda.
- Experimental validation of predicted miRNA-target interactions using luciferase reporter assays.
- Overexpression of miR-124 in mouse embryonic neural progenitor cells (eNPCs) followed by analysis of Fmr1, Cdk4, and cyclin D1 levels and cell proliferation.
Main Results:
- Identified 61 candidate miRNAs targeting Fmr1 predicted by all three computational tools.
- Experimentally verified 5 miRNAs (miR-23a, miR-32, miR-124, miR-335-5p, miR-350) targeting Fmr1.
- Overexpression of miR-124 significantly reduced Fmr1 levels and increased Cdk4 and cyclin D1 expression in eNPCs, promoting proliferation.
Conclusions:
- miR-124 directly targets and inhibits Fmr1 expression.
- miR-124 promotes mouse embryonic stem cell proliferation by upregulating Cdk4 and cyclin D1.
- These findings highlight miR-124 as a potential therapeutic target for conditions involving Fmr1 dysregulation and impaired cell proliferation.
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