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Redox imbalance of the endothelial cells (ECs) plays a causative role in a variety of cardiovascular diseases. In order to better understand the molecular mechanisms of the endothelial response to oxidative stress, the involvement of circular RNAs (circRNAs) was investigated. CircRNAs are RNA species generated by a "back-splicing" event, which is the covalent linking of the 3′- and 5′-ends of exons. Bioinformatics analysis of the transcriptomic landscape of human ECs exposed to H2O2 allowed us to identify a subset of highly expressed circRNAs compared to their linear RNA counterparts, suggesting a potential biological relevance. Specifically, circular Ankyrin Repeat Domain 12 (circANKRD12), derived from the junction of exon 2 and exon 8 of the ANKRD12 gene (hsa_circ_0000826), was significantly induced in H2O2-treated ECs. Conversely, the linear RNA isoform of ANKRD12 was not modulated. An increased circular-to-linear ratio of ANKRD12 was also observed in cultured ECs exposed to hypoxia and in skeletal muscle biopsies of patients affected by critical limb ischemia (CLI), two conditions associated with redox imbalance and oxidative stress. The functional relevance of circANKRD12 was shown by the inhibition of EC formation of capillary-like structures upon silencing of the circular but not of the linear isoform of ANKRD12. Bioinformatics analysis of the circANKRD12–miRNA–mRNA regulatory network in H2O2-treated ECs identified the enrichment of the p53 and Foxo signaling pathways, both crucial in the cellular response to redox imbalance. In keeping with the antiproliferative action of the p53 pathway, circANKRD12 silencing inhibited EC proliferation. In conclusion, this study indicates circANKRD12 as an important player in ECs exposed to oxidative stress. [ABSTRACT FROM AUTHOR]

Circular RNAs are generated by back‐splicing of precursor‐mRNAs. Although they have been known for many years, only recently we have started to appreciate their widespread expression and their regulatory functions in a variety of biological processes. Not surprisingly, circular RNA dysregulation and participation in the pathogenic mechanisms have started to emerge in many instances, including cardiovascular diseases. Detection, differential expression analysis and validation are the three critical points for the characterization of any RNA, and circular RNAs are no exception. Their characteristics, however, generate several problems that are yet to be completely addressed, and literature still lacks comprehensive definitions of well‐defined best practices. We present a map of the current knowledge regarding circular RNAs and the critical issues limiting our understanding of their regulation and function. The goal was to provide the readers with the tools to critically decide which of the many approaches available is most suitable to their experimental plan. Although particularly focused on cardiovascular diseases, most critical issues concerning circular RNAs are common to many other fields of investigation. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3′- and 5′-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients. [ABSTRACT FROM AUTHOR]