The long non-coding RNA ANRIL, antisense to the CDKN2B locus, was identified in familial melanoma patients, and contains multiple disease-associated polymorphisms. Divergent reports have suggested both concordant and discordant expression of CDKN2A or CDKN2B and ANRIL in different tissues and cell types. In view of these conflicting studies, and given the recurrent involvement of the CDKN2A gene (which encodes two tumor suppressor proteins p16^INK4 and p14^ARF) in melanoma, we attempted to analyze expression and characterize the isoforms of ANRIL in melanoma cell lines.

Gene expression analyses using quantitative RT-PCR revealed that CDKN2A and ANRIL expression are positively correlated in a panel of melanoma cell lines. We confirmed p16^INK4a mRNA expression in several cell lines in which p16^INK4a protein is not detectable, ruling out transcriptional suppression of p16^INK4a by ANRIL. This suggested that an epigenetic mechanism may regulate p16^INK4a mRNA function post-transcriptionally.

In addition to linear isoforms, we also identified circular forms of ANRIL (circANRIL) in these cell lines using quantitative RT-PCR. Further characterisation of circular isoforms of ANRIL in melanoma cell lines using outward-facing primers against each exon, followed by cloning and sequencing, revealed the existence of an assortment of circular isoforms with varying lengths, exon-exon junctions and exon combinations. This reveals the dynamic nature of the locus and adds complexity to investigating the function of ANRIL in melanoma.

We further found variability in the localization pattern of these isoforms with linear ANRIL enriched in the nucleus and the circular isoforms enriched in the cytoplasm. This variability in localization pattern suggests the hypothesis that ANRIL may have dual functions in melanoma.