Exploratory Circular RNA Profiling in Adrenocortical Tumors

SIMPLE SUMMARY: The histological differential diagnosis of adrenocortical adenoma and carcinoma is difficult and requires great expertise. Measures taken towards the distinction of adrenal tumors are of paramount importance. The non-coding circular RNAs (circRNAs) were shown to be expressed in a tissue and tumor specific manner. CircRNAs are investigated as a useful adjunct to the differential diagnosis of benign and malignant tumors of several organs, but they have not been investigated in adrenocortical tumors yet. Here, we have performed circRNA profiling in adrenocortical tumors by next-generation sequencing to detect already known and de novo circRNAs. Out of the five most differentially expressed circRNAs, circPHC3 could be confirmed by TaqMan RT-qPCR to be overexpressed in carcinoma and adenoma vs. healthy tissues in an independent validation cohort. ABSTRACT: Differentiation of adrenocortical adenoma (ACA) and carcinoma (ACC) is often challenging even in the histological analysis. Circular RNAs (circRNAs) belonging to the group of non-coding RNAs have been implicated as relevant factors in tumorigenesis. Our aim was to explore circRNA expression profiles in adrenocortical tumors by next-generation sequencing followed by RT-qPCR validation. Archived FFPE (formalin-fixed, paraffin embedded) including 8 ACC, 8 ACA and 8 normal adrenal cortices (NAC) were used in the discovery cohort. For de novo and known circRNA expression profiling, a next-generation sequencing platform was used. CIRI2, CircExplorer2, AutoCirc bioinformatics tools were used for the discovery of circRNAs. The top five most differentially circRNAs were measured by RT-qPCR in an independent validation cohort (10 ACC, 8 ACA, 8 NAC). In silico predicted, interacting microRNAs potentially sponged by differentially expressed circRNAs were studied by individual RT-qPCR assays. We focused on overexpressed circRNAs here. Significantly differentially expressed circRNAs have been revealed between the cohorts by NGS. Only circPHC3 could be confirmed to be significantly overexpressed in ACC, ACA vs. NAC samples by RT-qPCR. We could not observe microRNA expression changes fully corresponding to our sponging hypothesis. To the best of our knowledge, our study is the first to investigate circRNAs in adrenocortical tumors. Further studies are warranted to explore their biological and diagnostic relevance.