Osteopontin primary transcript generates exonic circRNA molecules by back splicing in tumor cell lines

Osteopontin (OPN) is a phosphoglycoprotein overexpressed in several human cancers and regulated by complex transcriptional and post-transcriptional mechanisms, including alternative splicing. The OPN transcript generates at least five splice variants (OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5), associated with distinct biological roles in tumor progression. Recently, circular RNAs (circRNAs) generated through back-splicing have emerged as regulators of gene expression in cancer. However, circRNAs originating from the OPN transcript have not yet been reported. Divergent primer pairs were designed across several OPN-derived exon-exon junction regions (ODEJRs) to selectively target exonic circRNAs generated by back-splicing. Conventional RT-PCR and RT-qPCR analyses using cDNA samples from distinct tumor cell lines (TPC1, 8505c, Caco-2, HT-29, HCT-116, RS4;11, Jurkat, and LNCaP) revealed the amplification of ODEJRs encompassing OPN coding exons 2 to 7, consistent with back-splicing events. Moreover, the expression profiles of these back-spliced ODEJRs varied across the tested cell lines, suggesting a cell-type-specific exonic OPN-derived circRNA signature. Collectively, our findings provide the first experimental evidence of circRNA molecules derived from the OPN primary transcript in tumor cells, expanding the known complexity of OPN post-transcriptional regulation.