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. 2021 Sep 17:9:e12147.
doi: 10.7717/peerj.12147. eCollection 2021.

Exosomal hsa-miR-21-5p is a biomarker for breast cancer diagnosis

Min Liu  1   2   3 , Fei Mo  2   3 , Xiaohan Song  3 , Yun He  2 , Yan Yuan  3 , Jiaoyan Yan  3 , Ye Yang  3 , Jian Huang  2 , Shu Zhang  2   3
Affiliations

Exosomal hsa-miR-21-5p is a biomarker for breast cancer diagnosis

Min Liu et al. PeerJ. .

Abstract

Purpose: Breast cancer (BC) is characterized by concealed onset, delayed diagnosis, and high fatality rates making it particularly dangerous to patients' health. The purpose of this study was to use comprehensive bioinformatics analysis and experimental verification to find a new biomarker for BC diagnosis.

Methods: We comprehensively analyzed microRNA (miRNA) and mRNA expression profiles from the Gene Expression Omnibus (GEO) and screened out differentially-expressed (DE) miRNAs and mRNAs. We used the miRNet website to predict potential DE-miRNA target genes. Using the Database for Annotation, Visualization and Integrated Discovery (DAVID), we performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on overlapping potential target genes and DE-mRNAs. The protein-protein interaction (PPI) network was then established. The miRNA-mRNA regulatory network was constructed using Cytoscape and the analysis results were visualized. We verified the expression of the most up-regulated DE-miRNA using reverse transcription and a quantitative polymerase chain reaction in BC tissue. The diagnostic value of the most up-regulated DE-miRNA was further explored across three levels: plasma-derived exosomes, cells, and cell exosomes.

Results: Our comprehensive bioinformatics analysis and experimental results showed that hsa-miR-21-5p was significantly up-regulated in BC tissue, cells, and exosomes. Our results also revealed that tumor-derived hsa-miR-21-5p could be packaged in exosomes and released into peripheral blood. Additionally, when evaluating the diagnostic value of plasma exosomal hsa-miR-21-5p, we found that it was significantly up-regulated in BC patients. Receiver operating characteristic (ROC) analysis also confirmed that hsa-miR-21-5p could effectively distinguish healthy people from BC patients. The sensitivity and specificity were 86.7% and 93.3%, respectively.

Conclusion: This study's results showed that plasma exosomal hsa-miR-21-5p could be used as a biomarker for BC diagnosis.

Keywords: Bioinformatics; Biomarker; Breast cancer; Diagnosis; Exosome; microRNA.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Detailed process of the proposed method. PPI, protein–protein interaction.
Figure 2
Figure 2. DE-miRNAs and DE-mRNAs were identified from GSE97811 and GSE29044, respectively.
Red represents up-regulation; green represents down-regulation (A) Volcanic map of miRNAs in GSE97811. (B) Volcanic map of mRNAs in GSE29044.
Figure 3
Figure 3. Venn diagrams of target mRNAs and DE-mRNAs. Target, predicted target mRNAs; Diff, DE-mRNAs obtained by GSE29044.
Figure 4
Figure 4. The results of GO and KEGG analyses of 52 overlapping mRNAs.
(A) The top 10 GO terms in the BP results of 52 overlapping mRNAs. (B) The top 10 GO terms in the MF results of 52 overlapping mRNAs. (C) The top 10 GO terms in the CC results of 52 overlapping mRNAs. (D) The results of KEGG pathway enrichment analysis of 52 overlapping mRNAs.
Figure 5
Figure 5. Construction of PPI and miRNA-mRNA networks. PPI: protein–protein interaction.
(A) PPI network of 52 overlapping mRNAs. (B) The miRNA-mRNA network constructed by 15 hub genes and DE-miRNAs. (C) The identified degree ranks the top 10 miRNA-mRNA networks.
Figure 6
Figure 6. The expression of hsa-miR-21-5p was significantly up-regulated in breast cancer tissues and breast cancer cells.
(A) The expression of hsa-miR-21-5p in 30 pairs of breast cancer tissues and corresponding adjacent tissues. (B) The expression of hsa-miR-21-5p in GSE97811. (C) The expression of hsa-miR-21-5p in breast cancer cell lines. ∗P < 0.05 vs. a MCF10A cell group; #P < 0.05 vs. a MCF10A exosome group.
Figure 7
Figure 7. Extraction and identification of exosomes.
(A) TEM characteristics of exosomes. (B) The distribution of exosome particles. (C) The results of western blotting characterization of exosomes.
Figure 8
Figure 8. Elevated plasma hsa-miR-21-5p was tumor-derived after packaging into exosomes.
(A) Expression of hsa-miR-21-5p in plasma exosomes of 30 patients with breast cancer before and after surgery. (B) The expression of hsa-miR-21-5p in plasma exosomes of patients with breast cancer was positively correlated with it in breast cancer tissues. (C) The expression of hsa-miR-21-5p in MDA-MB-231 and MCF7 exosomes varied with the duration of culture, ∗P < 0.05.
Figure 9
Figure 9. The expression of the exosome hsa-miR-21-5p of plasma and its diagnostic value.
(A) hsa-miR-21-5p in plasma exosomes of other cancer patients, HC, healthy control;BC, breast cancer; LIHC, liver hepatocellular carcinoma; LCa, lung cancer; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; OV, ovarian serous cystadenocarcinoma. (B) An ROC curve was used to analyze the sensitivity and specificity of hsa-miR-21-5p in the diagnosis of breast cancer. ∗P < 0.05; ns, no significance.
Figure 10
Figure 10. The expression of TGFβR3 and EGFR in breast cancer tissues.
(A) The expression of TGFβR3 in breast cancer patients of different stages. ∗P < 0.05 vs. a Normal group. (B) The expression of EGFR in breast cancer tissues. (C) The expression of TGFβR3 and EGFR in the tissues of breast cancer patients.
Figure 11
Figure 11. Kaplan–Meier curve analysis of potential target genes, TGFβR3 and EGFR, of hsa-miR-21-5p.
(A) A Kaplan–Meier curve was used to analyze the relationship between TGFβR3 and OS in patients with breast cancer. (B) A Kaplan–Meier curve was used to analyze the relationship between EGFR and OS in patients with breast cancer.
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