Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 28;49(4):562-577.
doi: 10.11817/j.issn.1672-7347.2024.230343.

Identification of ceRNA networks in type H and L vascular endothelial cells through integrated bioinformatics methods

[Article in English, Chinese]
Zhi Liu  1 , Zhe Ruan  2 , Haitao Long  3 , Ruibo Zhao  3 , Yong Zhu  3 , Zhangyuan Lin  3 , Peng Chen  4   5 , Shushan Zhao  6   7
Affiliations

Identification of ceRNA networks in type H and L vascular endothelial cells through integrated bioinformatics methods

[Article in English, Chinese]
Zhi Liu et al. Zhong Nan Da Xue Xue Bao Yi Xue Ban. .

Abstract
in English, Chinese, Chinese

Objectives: Type H blood vessels are a subtype of bone-specific microvessels (CD31hiEmcnhi) that play an important regulatory role in the coupling of angiogenesis and osteogenesis. Despite reports on the distinct roles of type H and L vessels under physiological and pathological bone conditions, their genetic differences remain to be elucidated. This study aims to construct a competitive endogenous RNA (ceRNA) network of key gene for differencial expression (DE) in type H and L vascular endothelial cells (ECs) through integrated bioinformatic methods.

Methods: We downloaded relevant raw data from the ArrayExpress and the Gene Expression Omnibus (GEO) database and used the Limma R-Bioconductor package to screen for DE lncRNAs, DE miRNAs, and DE mRNAs between type H and L vascular ECs. A total ceRNA network was constructed based on their interactions, followed by refinement using protein-protein interaction (PPI) networks to select upregulated and downregulated key genes. Enrichment analysis was performed on these key genes. Random validation was conducted using flow cytometry and real-time RT-PCR.

Results: A total of 1 761 DE mRNAs, 187 DE lncRNAs, and 159 DE miRNAs were identified, and a comprehensive ceRNA network was constructed based on their interactions. Six upregulated (Itga5, Kdr, Tjp1, Pecam1, Cdh5, and Ptk2) and 2 downregulated (Csf1r and Il10) key genes were selected via PPI network to construct a subnetwork of ceRNAs related to these key genes. Upregulated key genes were mainly enriched in negative regulation of angiogenesis and vascular apoptosis. Results from flow cytometry and real-time RT-PCR were consistent with bioinformatics analysis.

Conclusions: This study proposes a ceRNA network associated with upregulated and downregulated type H and L vascular ECs based on selected key genes, providing new insights into the regulatory mechanisms of type H and L vascular ECs in bone metabolism.

目的: H型血管是一种骨特异性微血管亚型(CD31hiEmcnhi)，在成血管-成骨耦联机制中具有重要的调节作用。研究报道H和L型血管在骨骼生理和病理条件下发挥不同的作用，它们之间的遗传差异仍有待阐明。本研究旨在通过整合的生物信息学方法构建H和L型血管内皮细胞中关键差异表达(differential expression，DE)基因的竞争性内源RNA(competitive endogenous RNA，ceRNA)网络。方法: 从ArrayExpress和基因表达综合(Gene Expression Omnibus，GEO)数据库中下载相关原始数据，通过Limma R-Bioconductor包筛选H和L型血管间的DE lncRNAs、DE miRNAs和DE mRNAs，构建总ceRNA网络，随后根据蛋白质相互作用(protein-protein interaction，PPI)网络筛选出上调和下调的关键基因，以此精细化ceRNA网络并对关键基因进行富集分析。最后，通过流式细胞术和real time RT-PCR进行随机验证。结果: 共鉴定出1 761个DE mRNAs、187个DE lncRNAs和159个DE miRNAs，通过相互作用关系构建总ceRNA网络；通过PPI网络筛选出6个上调(Itga5、Kdr、Tjp1、Pecam1、Cdh5、Ptk2)和2个下调(Csf1r、Il10)的关键基因，并以此构建关键基因相关的ceRNA亚网络。上调的关键基因主要富集在血管生成与血管凋亡的负调控；流式细胞术和real-time RT-PCR的结果与生物信息学分析结果一致。结论: 本研究根据所筛选的关键基因提出上调和下调的H型和L型血管内皮细胞相关的ceRNA网络，为H型和L型血管内皮细胞在骨代谢中的调控机制提供了新见解。.

目的: H型血管是一种骨特异性微血管亚型(CD31 hiEmcn hi)，在成血管-成骨耦联机制中具有重要的调节作用。研究报道H和L型血管在骨骼生理和病理条件下发挥不同的作用，它们之间的遗传差异仍有待阐明。本研究旨在通过整合的生物信息学方法构建H和L型血管内皮细胞中关键差异表达(differential expression，DE)基因的竞争性内源RNA(competitive endogenous RNA，ceRNA)网络。

方法: 从ArrayExpress和基因表达综合(Gene Expression Omnibus，GEO)数据库中下载相关原始数据，通过Limma R-Bioconductor包筛选H和L型血管间的DE lncRNAs、DE miRNAs和DE mRNAs，构建总ceRNA网络，随后根据蛋白质相互作用(protein-protein interaction，PPI)网络筛选出上调和下调的关键基因，以此精细化ceRNA网络并对关键基因进行富集分析。最后，通过流式细胞术和real time RT-PCR进行随机验证。

结果: 共鉴定出1 761个DE mRNAs、187个DE lncRNAs和159个DE miRNAs，通过相互作用关系构建总ceRNA网络；通过PPI网络筛选出6个上调( Itga5、 Kdr、 Tjp1、 Pecam1、 Cdh5、 Ptk2)和2个下调( Csf1r、 Il10)的关键基因，并以此构建关键基因相关的ceRNA亚网络。上调的关键基因主要富集在血管生成与血管凋亡的负调控；流式细胞术和real-time RT-PCR的结果与生物信息学分析结果一致。

结论: 本研究根据所筛选的关键基因提出上调和下调的H型和L型血管内皮细胞相关的ceRNA网络，为H型和L型血管内皮细胞在骨代谢中的调控机制提供了新见解。

Keywords: bioinformatics; bone vessels; ceRNA network; type H vascular endothelial cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Flow chart of the research PPI: Protein-protein interaction; ceRNA: Competitive endogenous RNA.
Figure 2
Figure 2. Expression profiles of mRNAs, lncRNAs, and miRNAs A-C: Cluster analysis of DE mRNAs (A), DE lncRNAs (B), and DE miRNAs (C), respectively. The red color indicates increased expression, the blue color indicates decreased expression, and the black color indicates no significant change. D-F: Cluster analysis of DE mRNAs (D), DE lncRNAs (E), and DE miRNAs (F), respectively. DE: Differential expression; FC: Fold change.
Figure 3
Figure 3. GO enrichment analyses of DE mRNAs A: Top 10 enriched GO terms of the DE mRNAs. B: Chord diagram shows the top 10 enriched biological processes of DE mRNAs. C: Circos plot shows the top 5 enriched biological processes of DE mRNAs. GO: Gene Ontology; DE: Differential expression.
Figure 4
Figure 4. KEGG pathway enrichment analyses of DE mRNAs A: Top 45 enriched KEGG pathways of DE mRNAs. B: Chord diagram shows the top 10 enriched KEGG pathways of DE mRNAs. C: Circos plot shows the top 5 enriched biological processes of DE mRNAs. KEGG: Kyoto encyclopedia of genes and genomes; DE: Differential expression; FC: Fold change; ECM: Extracellular matrix; PI3K-Akt: Phosphatidylinositol 3-kinase/protein kinase B.
Figure 5
Figure 5. CeRNA network with the most differentially expressed A: Up regulated ceRNA network; B: Down regulated ceRNA network. Red represents the mRNAs, blue represents the lncRNAs, and green represents the miRNAs. CeRNA: Competitive endogenous RNA; lncRNA: Long non-coding RNAs.
Figure 6
Figure 6. Key genes-related ceRNA networks A: Upregulated key genes-related ceRNA network; B: Downregulated key genes-related ceRNA network. Red represents the mRNAs, blue represents the lncRNAs, and green represents the miRNAs. CeRNA: Competitive endogenous RNA.
Figure 7
Figure 7. Functional enrichment analysis of upregulated key genes
Figure 8
Figure 8. Validation of ceRNA network by flow cytometry and real-time RT-PCR A: Relative expression of Itga5, miR-1894-3P, and Gm53 in type H and type L vascular ECs; B: Relative expression of Cdh5, miR-1224-5P, and Gm11707 in type H and type L vascular ECs; C: Relative expression of Csf1r, miR-423-5P, and Gm16587 in type H and type L vascular ECs. ** P<0.01, *** P<0.001. APC: Allophycocyanin; CeRNA: Competitive endogenous RNA; EC: Endothelial cell.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis[J]. Cell, 2011, 146( 6): 873- 887. 10.1016/j.cell.2011.08.039. - DOI - PubMed
    1. Ding BS, Cao ZW, LIS R, et al. . Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis[J]. Nature, 2014, 505( 7481): 97- 102. 10.1038/nature12681. - DOI - PMC - PubMed
    1. Maes C. Role and regulation of vascularization processes in endochondral bones[J]. Calcif Tissue Int, 2013, 92( 4): 307- 323. 10.1007/s00223-012-9689-z. - DOI - PubMed
    1. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[J]. Nature, 2014, 507( 7492): 323- 328. 10.1038/nature13145. - DOI - PMC - PubMed
    1. Xie H, Cui Z, Wang L, et al. . PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis[J]. Nat Med, 2014, 20( 11): 1270- 1278. 10.1038/nm.3668. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources