[Differences in circRNA expression profiles between HER-2-positive breast cancer cells and normal mammary epithelial...

Objective: To investigate the differential expression profiles of circular RNA (circRNA) in human epidermal growth factor receptor 2 (HER-2) positive breast cancer cells and normal mammary epithelial cells, and to develop novel diagnostic and therapeutic markers for HER-2 positive breast cancer. Methods: Total RNA were extracted from HER-2 positive breast cancer cell SK-BR-3 and normal mammary epithelial cell MCF10A. RNA quality was detected using NanoDrop ND-1000. Rnase R was applied to remove linear RNA and enrich circRNAs. After amplification and reverse transcription into fluorescent complementary RNA (cRNA) using random primer, the labeled cRNAs were hybridized onto the Arraystar Human circRNA Arrays. The raw data were extracted and the acquired array images were subjected to quantile normalization followed by heat map and volcano plot analysis. The expression of circRNAs with large fold change was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in the differentially expressed circRNAs and circRNA-microRNA (miRNA) network was constructed. Results: The total RNA extracted from SK-BR-3 and MCF10A had high integrity and quality. The expression profiles of circRNA in SK-BR-3 and MCF10A cells were significantly different shown by fluorescent expression signals. Compared with MCF10A cells, there were 6 584 up-regulated circRNAs and 6254 down-regulated circRNAs in SK-BR-3 cells. There were 348 circRNAs with |log(2)FC|≥2, of which 153 were up-regulated and 195 were down-regulated. Moreover, 8 circRNAs with |log(2)FC|>5. Among them, 5 were up-regulated in SK-BR-3 cells, including hsa_circRNA_074595 (|log(2)FC|=7.84), hsa_circRNA_074598 (|log(2)FC|=6.50), hsa_circRNA_085362 (|log(2)FC|=5.86), hsa_circRNA_101379 (|log(2)FC|=5.71) and hsa_circRNA_406683 (|log(2)FC|=5.34); as well as 3 were down-regulated, including hsa_circRNA_021714 (|log(2)FC|=5.46), hsa_circRNA_100777 (|log(2)FC|=5.40), and hsa_circRNA_100796 (|log(2)FC|=5.03). The expression levels of hsa_circRNA_074595, hsa_circRNA_074598 and hsa_circRNA_100777 were further validated by RT-qPCR in consistent with the results of microarray. GO analysis showed that differentially expressed circRNAs were significantly enriched in the biological process of heart development (P<0.001), cellular component in the cell adhesion-related components (P<0.001), molecular function in protein serine/threonine kinase activity (P<0.001). KEGG analysis revealed that differentially expressed circRNAs were significantly enriched in the PI3K-Akt signaling pathway. Conclusions: The expression profile of circRNA in HER-2 positive breast cancer cells is significantly different from that in normal mammary epithelial cells. The differentially expressed circRNAs may be served as potential diagnostic or therapeutic targets for HER-2 positive breast cancer.