Microarray Expression Profile and Analysis of Circular RNA Regulatory Network in Pulmonary Thromboembolism

Abstract

Background: Pulmonary thromboembolism (PTE) is a common disease which may be a serious condition and has high mortality. Recently, it has been shown that circRNAs play an important role in the development of various diseases, including thromboembolic disease. However, circRNAs expression profiling is not clear in PTE, this study aims to identify the circRNAs expressed in PTE and to elucidate their possible role in pathophysiology of PTE.

Methods: A total of 5 patients with CTPA-confirmed PTE and 5 healthy controls were recruited for the present study. The circRNAs expression profile was analyzed by microarray.

Results: In total, 256 differentially expressed circRNAs (up 142, down114) and 1162 mRNA (up 446, down 716) were summarized by analyzing the circRNAs microarray data. The top 3 up-regulated and 3 down-regulated circRNAs were validated by Real-Time Polymerase Chain Reaction (qRT-PCR). Two differentially expressed circRNAs (hsa_circ_0000891, hsa_circ_0043506) were selected for further analysis. Finally, we construct a circRNA-miRNA-mRNA ceRNA network with a bioinformatic prediction tool. Pathway analysis shows that the enriched mRNAs targets take part in Protein processing in endoplasmic reticulum, Systemic lupus erythematosus, Endocytosis, Spliceosome, HTLV-I infection and Ubiquitin mediated proteolysis.

Conclusion: Our findings indicated that aberrantly expressed circRNAs (hsa_circ_0000891, hsa_circ_0043506) may be involved in the development of PTE.

Keywords: circRNA; circRNA-miRNA-mRNA network; functional enrichment; gene; pulmonary thromboembolism.

Figure 1

Microarray analysis of differential expression of circRNAs, miRNAs and mRNAs in two groups. Hierarchical clustering analysis of differentially expressed circRNAs (|logFC| ≥2.0, P < 0.05) (A), miRNAs (|logFC| ≥0.5, P < 0.05) (B) and mRNAs (|logFC| ≥1.5, P < 0.05) (C) between PTE group and HC group; each group included five individuals. “Red” indicates high relative expression, and “Blue” indicates low relative expression. Volcano plot showing the differentially expressed circRNAs (D), miRNAs (E) and mRNAs (F) in the two groups, with “Red” and “Green” points representing the up-regulated and down-regulated transcripts, respectively.

Figure 2

GO and KEGG analyses of differentially expressed mRNAs. GO annotation of down-regulated (A) and up-regulated (B) mRNAs. KEGG pathway enrichment analyses for down-regulated (C) and up-regulated (D) mRNAs.

Figure 3

The principle component analysis (PCA) on the microarray data. (A) PCA was showed on the differentially expressed circRNAs. (B) PCA plot of the differentially expressed mRNAs.

Figure 4

Validation of candidate circRNAs using qRT-PCR. (A and B) The relative expression of has_circ_0043506 and has_circ_0000891 was confirmed to be up-regulated. (C) The relative expression of hsa_circ_0059324 had a down trend, but not statistically significant.

Figure 5

GO and KEGG analyses were performed for the predicted target genes of differentially expressed circRNA. (A) GO enrichment for targeted mRNAs. (B) KEGG pathway enrichment for targeted mRNAs.

Figure 6

A ceRNA network of circRNA-miRNA-mRNA interactions. Diamond, circRNA; V, miRNA; round, mRNA; red, up-regulated; blue, down-regulated.