MicroRNAs (miRNAs) are B22 nt non-coding RNAs that typically bind to the 3 0 UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR-200 family (miR-200a, -200b, -200c, -141 and -429) MicroRNAs (miRNA) are a class of $22nt noncoding RNAs that regulate gene expression post-transcriptionally.1 It is widely accepted that miRNA expression is broadly altered in most forms of cancer, 2 and loss of miR-200 family (miR200a, -200b, -200c, -141 and-429) expression has been reported in several types of advanced carcinoma, including bladder cancer. 3,4 Recently, several independent reports have implicated miR-200 and miR-205 in epithelial to mesenchymal transition (EMT), an important event in tumorigenesis associated with a decrease in E-cadherin levels, loss of cell adhesion and subsequent tumor invasion and metastasis. 5-9The miR-200s and miR-205 are key determinators of the epithelial phenotype by directly targeting ZEB1 (also known as TCF8) and ZEB2 (also known as SIP-1), which are transcriptional repressors of E-cadherin (CDH1). Loss of miR-200 expression thus leads to accumulation of ZEB1 and ZEB2, which is sufficient to silence CDH1 and promote EMT and tumor invasion. Interestingly, ZEB1 has been shown to act as a transcriptional repressor of miR-200c and miR-141 by directly interacting with E-and/or Z-box motifs in the miR-200c-141 promoter through a double-negative feedback mechanism. 8,9 However, the mechanisms regulating miR-200 and miR-205 expression remain otherwise largely uncharacterized. A recent study also concluded that ZEB1 is necessary for development and maintenance of stemness in cancer cells, thereby

BackgroundMicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.MethodsTaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.ResultsMiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.ConclusionsMiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.