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Identification of potential microRNA-target pairs associated with osteopetrosis by deep sequencing, iTRAQ proteomics
Minglin Ou1, Xiaoqing Zhang1, Yong Dai1
1Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China.
Abstract:
MicroRNAs aberrantly express in many human diseases including some metabolic bone disorders. They have been found to be associated with osteoclast differentiation and function, which makes them attractive candidates for the therapy of bone. However, the potential clinical application of microRNAs in therapeutics rests heavily upon our in-depth understanding of microRNAs and their targets. To identify potential microRNA-target pairs associated with osteopetrosis, we performed a system approach including deep sequencing, iTRAQ quantitative proteomics, and bioinformatics in the peripheral blood mononuclear cells (PBMCs) taken from patients with osteopetrosis and health donors. Notably, 123 differently expressed microRNAs, 173 differently expressed proteins, and 117 computationally predicted microRNA-target pairs with reciprocally expressed level in PBMCs were found in the two sample groups. Functional annotation identified that the microRNA-target pairs were involved in cell growth, differentiation, cellular signaling network, and the network highlighted the microRNA-target pair of has-miR-320a and ADP ribosylation factor 1 (Arf1) potentially associated with CLCN7 mutations in osteopetrosis. The pair of has-miR-320a and Arf1 was further verified by real-time PCR, western blot, and the interaction between has-miR-320a and its targeted sequence on the Arf1 mRNAs was confirmed by luciferase assay. Collectively, the present study established a new system approach for the investigation of microRNAs, and the microRNA-target pairs, particular has-miR-320a and Arf1, may have important roles in osteopetrosis.
Insights
MicroRNAs are key in bone diseases like osteopetrosis. This study identified has-miR-320a and Arf1 as a potential microRNA-target pair involved in osteopetrosis pathogenesis.
Area of Science:
- Genomics and Molecular Biology
- Biochemistry
- Cell Biology
Background:
- MicroRNAs (miRNAs) are implicated in human diseases, including metabolic bone disorders.
- Their role in osteoclast differentiation and function suggests therapeutic potential for bone conditions.
- Understanding miRNA-target interactions is crucial for developing miRNA-based therapies.
Purpose of the Study:
- To identify novel microRNA-target pairs associated with osteopetrosis using a systems biology approach.
- To investigate the potential role of these pairs in the pathogenesis of osteopetrosis.
- To validate a specific miRNA-target interaction linked to osteopetrosis.
Main Methods:
- Deep sequencing and iTRAQ quantitative proteomics were employed on peripheral blood mononuclear cells (PBMCs) from osteopetrosis patients and healthy donors.
- Bioinformatic analysis was used to predict and identify differentially expressed miRNAs and proteins with reciprocal expression patterns.
- Experimental validation included real-time PCR, western blot, and luciferase assays to confirm miRNA-mRNA interactions.
Main Results:
- The study identified 123 differentially expressed miRNAs, 173 differentially expressed proteins, and 117 predicted miRNA-target pairs in PBMCs.
- Functional annotation revealed that these pairs are involved in cell growth, differentiation, and cellular signaling networks.
- The specific pair of has-miR-320a and ADP ribosylation factor 1 (Arf1) was highlighted and experimentally validated, showing a direct interaction and potential association with CLCN7 mutations in osteopetrosis.
Conclusions:
- A novel systems approach was established for investigating microRNAs and their targets in disease.
- The identified miRNA-target pairs, particularly has-miR-320a and Arf1, may play significant roles in the pathophysiology of osteopetrosis.
- This finding provides a foundation for exploring has-miR-320a and Arf1 as potential therapeutic targets for osteopetrosis.
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