Towards Functionally Characterising the Long Non-coding RNA circANRIL in Human Melanoma

Abstract

ANRIL is a long noncoding RNA transcribed antisense to the CDKN2A/B locus. This locus encodes three potent tumour suppressor proteins whose activity is lost in nearly 50% of melanomas. ANRIL is suggested to interact with this locus although the exact nature of the association remains poorly understood. In the literature ANRIL is mainly suggested to be a negative regulator of the CDKN2A/B locus via recruitment of polycomb repressor complexes. Recent studies have found ANRIL to exist in multiple circular isoforms in addition to its linear counterparts. Here, we have analysed the expression and investigated the functionality of circANRILin a melanoma cell line expressing the p16INK4A mRNA but lacking the p16 protein. We identified a large number of circANRILisoforms by systematically amplifying them using outward-facing primers specific for multiple exons. Characterisation of these isoforms will enable us to further analyse their functions, demonstrates the complexity of this locus and points to a complex regulatory network for p16INK4A expression. We investigated the sub-cellular localisation of circANRIL transcripts and found that they predominantly localise to the cytoplasm, which is distinct to the nuclear localisation of the linear isoforms. This suggests the possibility of different mechanisms of action for the two states. To gain insight into the potential role of circANRIL in the cytoplasm, we subjected cell extracts to density gradient centrifugation, examined the A260 profile of RNA followed by RT-PCR forcircANRIL and found it mainly to sediment in the same fractions as monosomes. It suggests that circANRIL may associate with supramolecular complexes. Together, these data suggest that circANRIL may exert its functions as a post-transcriptional regulator of p16INK4A. Recent studies have demonstrated the induction of ANRIL in a NF-κB-dependent manner in response to various inflammatory stimuli. We established methods to induce stress in the cells by inflammatory stimuli (stimulation with lipopolysaccharide) and measure the expression of cytokines, and of linear and circANRIL. Despite an upregulation in IL-6 and IL-8 in both cell lines, we found that the response in two melanoma cells lines for ANRIL was different. The general trend in NZM37 cells indicated an upregulation of circANRIL while linear ANRIL remains unchanged. This trend was not detected in NZM7 cells. This finding suggests the heterogeneity of these cell lines in response to inflammatory stimulation.