Sanger sequencing chromatograms.

Circular RNAs (circRNAs) constitute a large class of non-coding RNAs with gene regulatory capabilities, mainly through microRNA binding, a mechanism that has been linked to cancer development. The circRNA ciRS-7 (also known as CDR1as) is an interesting candidate as it harbors over 60 binding sites for miR-7, which is known to have tumor-suppressing properties. Here, we investigated the spatial expression patterns and epigenetic regulation of ciRS-7 across nine different adenocarcinomas originating from the colon, pancreas, ovary, endometrium, breast, stomach, bile duct, lung, and prostate. The study included primary patient samples and 18 different cell lines. ciRS-7 expression was analyzed using Reverse Transcription-quantitative PCR (RT-qPCR), single molecule in situ hybridization, and Nanostring nCounter, while epigenetic modifications were examined through bisulfite sequencing, Sensitive Melting Analysis after Real Time – Methylation Specific PCR (SMART-MSP), and chromatin immunoprecipitation. The functional relevance of epigenetic modifications was examined using DNA methyltransferase and histone deacetylase inhibitors. Across all adenocarcinomas, ciRS-7 was absent in the cancer cells in most of the primary tumor specimens, except for the breast tumors, while being expressed in the tumor microenvironment (TME). In line with this, ciRS-7 was not detected in most of the cell lines. Moreover, we demonstrated that DNA methylation and H3K9 acetylation, but not H3K27 methylation, are important epigenetic modifications that impact ciRS-7 expression. In conclusion, our data show that ciRS-7 is mainly expressed in the TME and is regulated through DNA methylation and histone acetylation across all major types of adenocarcinomas.