Luminal breast cancer-specific circular RNAs uncovered by a novel tool for data analysis

Abstract

Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcript processing. We have exploited poly(A-) RNA-Seq data generated in our lab in MCF-7 breast cancer cells to define a compilation of exonic circRNAs more comprehensive than previously existing lists. Development of a novel computational tool, named CircHunter, allowed us to more accurately characterize circRNAs and to quantitatively evaluate their expression in publicly available RNA-Seq data from breast cancer cell lines and tumor tissues. We observed and confirmed, by ChIP analysis, that exons involved in circularization events display significantly higher levels of the histone post-transcriptional modification H3K36me3 than non-circularizing exons. This result has potential impact on circRNA biogenesis since H3K36me3 has been involved in alternative splicing mechanisms. By analyzing an Ago-HITS-CLIP dataset we also found that circularizing exons overlapped with an unexpectedly higher number of Ago binding sites than non-circularizing exons. Finally, we observed that a subset of MCF-7 circRNAs are specific to tumor versus normal tissue, while others can distinguish Luminal from other tumor subtypes, thus suggesting that circRNAs can be exploited as novel biomarkers and drug targets for breast cancer.

Prediction of circRNAs Based on the DNA Methylation-Mediated Feature Sponge Function in Breast Cancer

“…Our research reveals a complex DNA methylationmediated circRNA sponge regulatory mechanism (Supplementary Figure 7). Interestingly, four of the 10 Sponge circRNAs (hsa_circ_000582, hsa_circ_000691, hsa_circ_000877, hsa_circ_000690) were identical to the breast cancer-specific circRNAs from previous studies (Coscujuela Tarrero et al, 2018). Sponge circRNAs may lead to abnormal expression of important protein coding driver genes in breast cancer.…”

The circular RNome of primary breast cancer

“…Although a synthetic circRNA construct including an internal ribosome entry site (IRES) can be translated (Wang and Wang 2015), current literature (Jeck et al 2013;Guo et al 2014;Szabo and Salzman 2016;Liang et al 2017;Liu et al 2018;Wang et al 2018;Zeng et al 2018) describes mostly noncoding functions for circRNAs, for example, as miRNA sponge circCDR1 (Hansen et al 2013;Memczak et al 2013;Zhao et al 2015;Kristensen et al 2018) and circZNF91 (Guo et al 2014), whereas associative evidence was reported (Rybak-Wolf et al 2015) of evolutionarily conserved circRNAs between human and mouse. Further support that circRNAs may be specifically generated and regulated was derived from a study using (estrogen-stimulated) MCF-7 cells (Tarrero et al 2018), showing higher levels of H3K36me3 (posttranscriptional histone modification) and a higher number of Ago binding sites in circularizing exons.…”

“…Of these, 2259 circRNAs (49.9%) were also present in our catalog. In addition, 3271 circRNAs were reported in an MCF-7 breast cancer cell line panel (Tarrero et al 2018). In total, 922 circRNAs showed (increased) expression in estrogen-stimulated MCF-7 cells compared with cells cultured in hormone-deprived medium, of which 733 circRNAs (79.5%) were present in our list.…”

Analysis of circular RNA‑associated competing endogenous RNA network in breast cancer

“…The development of effective therapies for patients with BC requires the identification of therapeutic and prognostic targets. Recent studies have focused on the identification of differentially expressed non-coding RNAs, including long non-coding RNAs, miRNAs and circRNAs, by gene chip and high-throughput sequencing (44)(45)(46). It is essential to identify early clinical diagnostic markers in order to understand the molecular mechanism underlying, and facilitate the diagnosis of, BC.…”

“…RNA-seq is a common method for genome-wide analysis of circRNAs, though limited by the low reads that cover the specific splicing junction (52). In previous studies, Lan et al (53) investigated circRNA expression profiles via high-throughput RNA-seq in papillary thyroid carcinoma, while Coscujuela et al (45) used RNA-seq to profile and characterize circRNAs in luminal-like BC. Nair et al (44) created a comprehensive workflow termed Circ-Seq based on existing bioinformatics approaches to screen expressed circRNAs in BC subtypes.…”