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. 2021 Dec;10(12):5133-5149.
doi: 10.21037/tcr-21-1771.

Reconstruction and analysis of the aberrant lncRNA-miRNA-mRNA network based on competitive endogenous RNA in adenoid cystic carcinoma of the salivary gland

Yu-Fang Tang #  1   2   3 , Wen-Jie Wu #  1   2   3 , Jian-Yun Zhang  2   3   4 , Jie Zhang  1   2   3
Affiliations

Reconstruction and analysis of the aberrant lncRNA-miRNA-mRNA network based on competitive endogenous RNA in adenoid cystic carcinoma of the salivary gland

Yu-Fang Tang et al. Transl Cancer Res. 2021 Dec.

Abstract

Background: The aim of this work was to investigate the competing endogenous RNA (ceRNA) network in adenoid cystic carcinoma of the salivary gland (SACC).

Methods: Differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) between cancer tissues and normal salivary gland (NSG) in ACC were identified using data from the Gene Expression Omnibus (GEO) database. Functional annotation and pathway enrichment analysis of DEmRNAs were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The miRNAs that are targeted by lncRNAs were predicted using miRanda and PITA, while the target mRNAs of miRNAs were retrieved from miRanda, miRWalk, and TargetScan. A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and then we constructed the lncRNA-miRNA-mRNA networks of ACC.

Results: Differentially expressed RNAs were identified in SACC. Upon comparing cancer tissues and NSG tissues, 103 upregulated and 52 downregulated lncRNAs and 745 upregulated and 866 downregulated mRNAs were identified in GSE88804; in addition, 39 upregulated and 43 downregulated miRNAs were identified in GSE117275. GO enrichment analyses revealed that the most relevant GO terms were regulation of transcription DNA-templated, transcription DNA-templated, and cell division. KEGG pathway enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the cell cycle, pathways in cancer, PI3K-Akt signaling pathway, breast cancer, and microRNAs in cancer. The PPI network consisted of 27 upregulated and 54 downregulated mRNAs. By constructing ceRNA network, NONHSAT251752.1-hsa-miR-6817-5p-NOTCH1, NONHSAT251752.1-hsa-miR-204-5p/hsa-miR-138-5p-CDK6 regulatory axises were identified and all genes in the network were verified by qRT-PCR.

Conclusions: The present study constructed ceRNA networks in SACC and provided a novel perspective of the molecular mechanisms for SACC.

Keywords: Salivary gland neoplasms; long non-coding RNA (lncRNA); microRNAs.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tcr-21-1771). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The experimental analysis flow chart.
Figure 2
Figure 2
Clustergram of DE-RNAs: Each column of the x-axis in the Clustergram represents a sample, and each row on the y-axis represents a different gene. Red represents a relatively high expression value of DEGs in the sample, while green represents a relatively low expression value of DEGs in the sample. Heatmap is built by the parameters of P<0.05 & FDR <0.05 & FC >2. (A) LncRNA-SACC-Normal; (B) miRNA-SACC-Normal; (C) mRNA-SACC-Normal. DE-RNAs, differentially expressed RNAs; DEGs, differentially expressed genes; lncRNA, long noncoding RNA; SACC, adenoid cystic carcinoma of the salivary gland.
Figure 3
Figure 3
GO analysis: the top 25 significant GO terms of the up-regulated and down-regulated genes. (A) Top 25 significant up-regulated genes in GO analysis; (B) top 25 significant down-regulated genes in GO analysis. GO, Gene Ontology.
Figure 4
Figure 4
Pathway analysis: The top 25 significant pathway analyses of up-regulated and down-regulated genes. (A) Top 25 significant up-regulated genes in pathway analysis; (B) top 25 significant down-regulated genes in pathway analysis.
Figure 5
Figure 5
LncRNA-miRNA-mRNA network: rectangles represent miRNAs, circles represent mRNAs, and triangles represent lncRNAs. Red represents up-regulated expression in cancer, and the green represents down-regulated expression in cancer. The size of the point represents the regulatory capacity of the mRNA; the larger the point is, the stronger the regulatory capacity. (A) LncRNA (up-regulated)-miRNA (down-regulated)-mRNA (up-regulated); (B) lncRNA (down-regulated)-miRNA (up-regulated)-mRNA (down-regulated).
Figure 6
Figure 6
PPI network: circle represents mRNAs. Red represents up-regulated mRNAs, green represents down-regulated mRNAs. The size of the point represents the regulatory capacity of the mRNA; the larger the point is, the stronger the regulatory capacity. PPI, protein-protein interaction.
Figure 7
Figure 7
Verification of the reliability of DE-RNAs by qRT-PCR: The up-regulated and down-regulated RNAs in bioinformatics analyses were detected by qRT-PCR in NSG and SACC tissues. (A) The up-regulated lncRNAs; (B) the down-regulated lncRNAs; (C) the up-regulated miRNAs; (D) The down-regulated miRNAs; (E) the up-regulated mRNAs; (F) The down-regulated mRNAs. *P<0.05, **P<0.01, ***P<0.001. NSG, normal salivary gland; SACC, adenoid cystic carcinoma of the salivary gland.
Figure 8
Figure 8
Expression of regulatory networks in NSG and SACC by qRT-PCR. (A) NONHSAT251752.1-hsa-miR-6817-5p-NOTCH1; (B) NONHSAT251752.1-hsa-miR-204-5p/hsa-miR-138-5p-CDK6; (C) NONHSAT232163.1/NONHSAT250051.1/NONHSAT192459.1-hsa-miR-34c-5p/hsa-miR-135a-3p-IGF1. *P<0.05, **P<0.01, ***P<0.001. NSG, normal salivary gland; SACC, adenoid cystic carcinoma of the salivary gland.
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