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Construction of dynamic ceRNA regulatory networks in osteogenesis during fracture healing based on transcriptomic
Shuhang Guo1,2, Shen Wang1,2, Shaoxun Yuan3
1Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China.
Abstract:
Fracture healing is a complex biological process. This study aimed to investigate the key molecules involved in fracture healing and their potential competing endogenous RNA (ceRNA) regulatory mechanisms within the first 28 days post-fracture using bioinformatics methods. The experiment was conducted on 15 adult male SD rats, with tibia callus tissue samples collected at days 0, 3, 7, 14, and 28 (n = 3) post-fracture. RNA-Seq was used for high-throughput transcriptome sequencing, followed by differential expression analysis to identify differentially expressed genes (DEGs), long non-coding RNA (DELs), and microRNA (DEMs) at different stages. Protein-protein interaction (PPI) networks were constructed using the STRING database and visualized with Cytoscape. GO and KEGG enrichment analyses were performed to explore potential biological mechanisms. miRNA-mRNA interactions were predicted using TargetScan, miRWalk, and miRDB, while RNA22 v2 was used for lncRNA-miRNA interactions. These interactions were integrated into ceRNA networks. Finally, qRT-PCR validated key molecules within the ceRNA network. We identified 4,997 DEGs, 315 DELs, and 89 DEMs at day 3; 5,087 DEGs, 300 DELs, and 84 DEMs at day 7; 3,073 DEGs, 235 DELs, and 68 DEMs at day 14; and 2,609 DEGs, 197 DELs, and 90 DEMs at day 28. Further analysis revealed hub osteogenic genes and their ceRNA regulatory networks at each time point. The networks consisted of 2 mRNAs, 3 miRNAs, and 9 lncRNAs at day 3; 2 mRNAs, 3 miRNAs, and 8 lncRNAs at days 7 and 14; and 1 mRNA, 3 miRNAs, and 10 lncRNAs at day 28. We validated two key lncRNAs (AABR07030366.1 and AABR07057997.1) along with their interacting miRNAs and mRNAs: rno-miR-9a-5p/Col9a1 (day 3), rno-miR-181c-5p/Comp (day 7), rno-miR-423-5p/Col1a1 (day 14), and rno-miR-185-5p/Ctsk (day 28). In summary, our study leveraged bioinformatics to construct ceRNA networks involved in osteogenesis post-fracture, offering insights into their dynamic regulatory role in healing and underlying molecular mechanisms.
Insights
This study reveals key molecular players and competing RNA networks regulating fracture healing in rats. Bioinformatics identified dynamic ceRNA networks crucial for osteogenesis and bone repair mechanisms.
Area of Science:
- Molecular Biology
- Bioinformatics
- Genomics
Background:
- Fracture healing is a complex biological process involving intricate molecular regulation.
- Understanding the competing endogenous RNA (ceRNA) mechanisms is crucial for elucidating fracture healing dynamics.
Purpose of the Study:
- To investigate key molecules and ceRNA regulatory networks in fracture healing within 28 days post-fracture.
- To identify differentially expressed genes, long non-coding RNAs, and microRNAs during distinct healing stages.
- To construct and validate ceRNA networks involved in osteogenesis during bone repair.
Main Methods:
- RNA sequencing (RNA-Seq) of rat tibia callus tissue at multiple time points (0, 3, 7, 14, 28 days).
- Bioinformatic analyses including differential expression, protein-protein interaction networks, GO/KEGG enrichment, and ceRNA network construction.
- Validation of key molecules within the ceRNA network using qRT-PCR.
Main Results:
- Identification of thousands of differentially expressed genes (DEGs), long non-coding RNAs (DELs), and microRNAs (DEMs) across healing stages.
- Construction of dynamic ceRNA networks comprising mRNAs, miRNAs, and lncRNAs at each time point.
- Validation of specific lncRNAs, miRNAs, and their target mRNAs (e.g., rno-miR-9a-5p/Col9a1 at day 3).
Conclusions:
- Bioinformatics approaches successfully constructed ceRNA networks crucial for osteogenesis during fracture healing.
- These networks highlight the dynamic regulatory roles of lncRNAs, miRNAs, and mRNAs in bone repair.
- The findings offer insights into the molecular mechanisms underlying fracture healing and potential therapeutic targets.
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