Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3′-end of an exon to the 5′-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.

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Circular RNAs (circRNAs) are a subset of noncoding RNAs previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display flanking sequences that suggest an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide open reading frame, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements.

miR-151a and its host gene, focal adhesion kinase, FAK, are located in a region of chromosome 8q that is frequently amplified in solid tumors, including lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and metastasis remains the major challenge in battling lung cancer mortality. Here, we demonstrate that miR-151a is overexpressed in non-small cell lung cancer (NSCLC) patient specimens, as compared to healthy lung. In addition, miR-151a overexpression promotes proliferation, epithelial-to-mesenchymal transition (EMT) and induces tumor cell migration and invasion of NSCLC cells. Blocking miR-151a expression using anti-miR-151a approaches significantly reduced NCSLC cell proliferative and motility potential. Furthermore, we determined that miR-151a significantly regulates E-cadherin expression. Finally, functional rescue experiments determined that overexpression of E-cadherin in miR-151a NSCLC cell lines potently repressed miR-151a-induced partial EMT and cell migration of NSCLC cells. In conclusion, our findings suggest that miR-151a functions as an oncomiR in NSCLC by targeting E-cadherin mRNA and inducing proliferation, migration and partial EMT.