Profiling and bioinformatics analyses reveal differential circular RNA expression in radioresistant esophageal cancer cells

Acquired radioresistance has been considered as one of the most important reasons causing treatment failure for esophageal cancer patients. In this study, we explored the expression patterns of circRNAs between radioresistant esophageal cancer cell line KYSE-150R and its parental cell line KYSE-150 with Arraystar Circular RNA Microarray to investigate the mechanisms of acquired radioresistance of esophageal cancer. Differentially expressed profiles of circRNAs in radioresistant esophageal cancer cells were observed and validated compared with the parental esophageal cancer cells, indicating possible involvement of these dysregulated circRNAs in the development of radiation resistance of esophageal cancer cells.

CircRNAs are discovered as new special kind of ubiquitous endogenous noncoding RNAs [23]. Recent evidences revealed that circRNAs can function as miRNA sponges and regulate parent gene expression to affect disease. Despite the potential importance of circRNAs reported in several types of cancer [24, 25], there is no reported studies on the functional roles of circRNAs in the radiation resistance of cancer. In this study, there were 57 circRNAs significantly upregulated and 17 circRNAs significantly downregulated in the KYSE-150R cell lines compared with KYSE-150, respectively. In which, circRNA_100385, circRNA_104983 and circRNA_001059 were upregulated with top magnitudes. CircRNA_101877, circRNA_102913, and circRNA_000695 were downregulated with top magnitudes. The expression pattern of these circRNAs were validated by qRT-PCR, and consistent results were observed. Our results indicated that the altered expression levels of circRNAs may be related to their involvement in the transcription level regulation on the radiation resistance of esophageal cancer cells. Aberrant expression of circRNAs has been linked to carcinogenesis and the malignant behavior of many different cancer. The abundance of a circRNA called hsa_circ_002059 has been reported to be significantly downregulated and suggested as a potential diagnostic marker for gastric cancer [26]. Qin M et al. [27] found that hsa_circ_0001649 may play a role in tumorigenesis and metastasis of hepatocellular carcinoma. Li et al. [28] reported that cir-ITCH might influence the expression level of ITCH and may be involved in the development of esophageal squamous cell carcinoma. However, no obvious changes of cir-ITCH was observed in this study, suggesting it may have no contribution to the radioresistance of esophageal cancer cells.

Despite the lack of knowledge of the exact functions of most circRNAs, we investigated the potential targets of these altered miRNAs. In a total, 57 upregulated circRNAs were identified to regulate the expression level of 120 microRNAs using the miRanda software, and 12 downregulated circRNAs were identified to regulate 36 microRNAs. According to the findings of Denzler et al. [29] low levels of circRNAs may not be sufficient to affect the target miRNAs. The circRNA-microRNA co-expression network analysis were conducted for our Top-5 circRNAs in this study. Two potential crucial circRNAs, circRNA_001059 and circRNA_000167 were identified to be influential on target miRNAs. CircRNAs were believed to negatively regulate miRNAs, and contribute substantially to the competing endogenous RNA (ceRNA) network. It has been reported that ciRS-7, as a circular miR-7 inhibitor, harbors more than 60 conventional miR-7 binding sites, which is far more than any known linear sponges [30]. Sex-determining region Y (SRY) was another identified miRNA sponge and functioned as a miR-138 sponge [31]. According to our results, we hypothesized that circRNA_001059 may act as an inhibitor of miRNA by binding several specific miRNAs, including miR-30c-1*, miR-30c-2*, miR-122*, miR-139-3p, miR-339-5p and miR-1912. Our results implied that it is worthwhile to further investigate these novel dysregulated circRNAs as microRNA sponges and their potential biological functions in the development of radiation resistance.

In this study, GO analysis and KEGG pathway annotation were conducted to investigate the functions of related microRNAs [32]. GO enrichment analysis revealed that target genes were involved in the regulation of crucial biological processes, indicating that regulating these genes in the cellular response is of great importance during the development of radioresistance. Among the upregulated pathways found in this study, phosphatidylinositol signaling pathway had been reported to be a key mediator of tumor cell responsiveness to radiation [33]. Phosphatidylinositol 3-kinase (PI3K)/Akt pathway accelerates the repair of DNA-DSB (DNA double-strand breaks), and consequently, its activation leads to therapy resistance [33]. Wnt signaling pathway which corresponds to downregulated circRNAs had also been reported to play predominant roles in radioresistance in Glioblastoma [34] and prostate cancer [35]. These results were also in line with the observations reported in our previous study [21].