Functional profiling of circular noncoding RNA circANKRD12 in cancer

ABSTRACT Introduction: Circular RNAs (circRNA) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional... [ view full abstract ]

ABSTRACT

Introduction: Circular RNAs (circRNA) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Circular RNAs are unusually stable and abundant with tissue or developmental stage specific expression pattern. These qualities mark them to be a potential biomarker or a specific therapeutic target.

Materials and methods: Cancer cell lines (breast, ovarian and lung) used for the study. Silencing of circAKRD12 and its linear RNA was done using RNAi method and validated by using Real Time PCR method. Gene expression analysis was done using RNA-Seq analysis using illumina HiSeq 4000 system. Functional and phenotypic analysis were done by cell proliferation assays, cell cycle analysis and 3D organotypic cells generation on circANKRD12 silenced cells.

Results and discussion. In this study, we identified and characterized a circular RNA named circANKRD12 derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12.This circANKRD12 was highly abundant in breast and ovarian cancers with two specific isoforms. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source gene mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provide evidence for its functional relevance through downstream regulation of several tumor invasion genes. Knockdown of this particular circANKRD12 affects several cell signaling pathways including cell cycle progression and immune modulatory pathways . We show that silencing of circANKRD12 induces a functionally relevant phenotypic change by significantly regulating cell cycle and increasing invasion and migration in cancer cells and shows an increased invasion through collagen gel in 3D spheroids.

These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression and can be a potential biomarker for either cancer diagnosis or prognosis.