A microarray expression profile and bioinformatic analysis of circular RNA in human esophageal carcinoma

Abstract

Background: Recent studies indicate that non-coding circular RNAs (circRNAs) are involved in the development of esophageal carcinoma (EC). This study aimed to identify differential expression of circRNAs in EC, which can provide potential biomarkers and therapeutic targets for EC treatment and improve the understanding of tumorigenesis mechanism.

Methods: First, samples (n=5) of EC tissues and adjacent normal tissue were sent for circRNA microarray detection, Second, further bioinformatic analysis was performed, including circRNA-microRNA (miRNA), co-expression network analysis, Spearman correlation test, and cancer-related circRNA-miRNA axis analysis. Finally, the expression of circRNA that our analysis predicted to be hub genes was verified in samples (n=15) of EC tissues and adjacent normal tissue by real-time polymerase chain reaction (RT-PCR).

Results: Microarray identified 102 upregulated and 67 significantly downregulated circRNAs were in EC patients' tumors relative to adjacent normal tissue. One upregulated circRNA (hsa_circRNA_401955) showed the most connection with MREs, therefore was regarded as the hub gene by the Spearman correlation test. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that four primary pathways (mRNA surveillance, cytoskeleton actin regulation, spliceosome, and the NOD-like receptor signaling pathway) were predicted in the hub circRNA's five connected miRNA response elements (MREs). Furthermore, cancer-related circRNA-miRNA axis analyses showed that hsa_circRNA_100375 and its four connected MREs participated in the cancer-related pathway. RT-PCR showed that hsa_circRNA_100375 and hsa_circRNA_401955 were significantly increased in the tumor tissues of EC patients.

Conclusions: Abnormal expression of circRNAs was involved in the tumorigenesis of EC. Key circRNAs, namely hsa_circRNA_401955 and hsa_circRNA_100375, may be as potential biomarkers and therapeutic targets for the treatment of EC.

Keywords: Esophageal carcinoma (EC); bioinformatic analysis; circular RNAs (circRNAs); microRNA sponge; microarray.

Figure 1

The distribution of circRNAs and distinguishable circRNAs identified through scatter plot and volcano plot. (A) The distribution of circRNAs shown by box plots in the 10 samples. After normalization, the distributions were nearly the same. (B) Left: a volcano plot of distinguished circRNAs. The dots above the top and the bottom green line revealed that the expression was more than 1.5-fold. Right: a volcano plot of circRNAs. The red point represents for 1.5-fold up and down regulated of circRNAs expression.

Figure 2

Profiling of circRNAs detected by microarray in esophageal carcinoma patients. (A) CircRNA microarray expression data among the 10 samples; (B) heap map of the differentially expressed circRNAs. The red represents high expression, while the green represents low expression.

Figure 3

The co-expression network of circRNA-miRNA. The network of significant differentially expressed circRNAs (fold change of more than 2.0) and its connected MREs was constructed by Cytocape. The up-regulated circ-RNA-miRNA coexpression network and the downregulated circ-RNA-miRNA co-expression network. MREs, miRNA response elements.

Figure 4

The co-expression network of the hub circRNA and its related circRNAs. The red nodes represent a positive correlation, the green nodes represent a negative correlation. The colors of the connected lines from black to blue represent a low to high value of the correlation coefficient.

Figure 5

The combined sites of the hub circRNA (hsa_circRNA_401955) and its 5 connected MREs. MREs, miRNA response elements.

Figure 6

KEGG pathway analysis of the hub circRNA’s (hsa_circRNA_401955) related with MREs. (A-E) KEGG pathway analysis of hsa-miR-1277-5p, hsa-miR-6504-5p, hsa-miR-3064-5p, hsa-miR-642a-5p, and has-miR-141-5p and their target genes predicted by TarBase, respectively. The primary pathway was cytoskeleton actin regulation in (A), spliceosome in (B) and (C), NOD-like receptor in (D), and mRNA surveillance in the (E) signaling pathway. P<0.05. KEGG, Kyoto Encyclopedia of Genes and Genomes; MREs, miRNA response elements.

Figure 7

KEGG pathway and protein analysis of hsa_circRNA_100375/hsa-miR-324-3p/hsa-miR-29a-5p/hsa-miR-485-3p/hsa-miR-149-5p axis. (A) The KEGG pathway showed the axis involved in the cancer-related pathway; (B) the combined sites of hsa_circRNA_100375 and its 4 cancer-related MREs (hsa-miR-324-3p/hsa-miR-29a-5p/hsa-miR-485-3p/hsa-miR-149-5p). (C) Certain hub proteins, such as FGFR1, PTGER2, WNT9B, WNT2B, SP1, CREBBP, IL-6, and DVL2, played key roles in the cancer-related pathway. KEGG, Kyoto Encyclopedia of Genes and Genomes. MREs, miRNA response elements. FGFR1, fibroblast growth factor receptor 1; PTGER2, prostaglandin E receptor 2; WNT9B, wingless-type MMTV integration site family, member 9B; WNT2B, wingless-type MMTV integration site family, member 2B; SP1, transcription factor Sp1; CREBBP, cAMP-response element binding protein; IL-6, interleukin-6; DVL2, disheveled 2.

Figure 8

The relative gene expression of the most differentially expressed circRNAs (hsa_circRNA_100375, hsa_circRNA_401955, the top 3 upregulated circRNAs, and the top 5 downregulated circRNAs). The tissue of 15 patients with esophageal carcinoma were analyzed by using RT-PCR. The normal adjacent tissue samples were used as the control. (A) The relative gene expression of hsa_circRNA_100375 and hsa_circRNA_401955. The results showed that the 2 circRNAs were significantly decreased in the tumor samples compared to the normal adjacent tissues (*, P<0.05). (B) The relative gene expression of the top 3 upregulated and top 5 downregulated circRNAs. The results showed that, compared with the control samples, the top 5 downregulated circRNAs (hsa_circRNA_102034, hsa_circRNA_100191, hsa_circRNA_101009, hsa_circRNA_037767, and hsa_circRNA_102459) were significantly decreased in the tumor tissues, while the top three upregulated circRNAs (hsa_circRNA_043621, hsa_circRNA_087961 and hsa_circRNA_404474) were significantly increased in the tumor tissues (*, P<0.05). RT-PCR, real-time polymerase chain reaction.