Definitive diagnosis of malignant pleural mesothelioma (MPM) is difficult and requires invasive tumour biopsy. MicroRNAs are proven to be dysregulated in MPM and have therapeutic potential. Circular RNAs (circRNAs) are non-coding competitive endogenous RNAs (ceRNAs) that interact with microRNAs as ‘sponges’ via direct binding, subsequently leading to their repression. CircRNAs are dysregulated in cancer and are cell-type specific, thermodynamically stable and highly conserved, thus, serve as potential blood-based biomarkers for detection of MPM. This study investigated circRNA gene expression patterns using MPM cell lines to identify potential candidates towards MPM diagnosis.
Total RNA was isolated from a series of 9 MPM and 2 normal mesothelial cell lines (n=3). Linear RNAs were removed by the addition of RNase R, to enrich circular RNAs. These were then amplified and labelled with fluorescence using a random priming method. Labelled circRNAs were hybridised onto Arraystar Human circRNA Array v2. Quantile normalisation of raw data was carried out using the R-software limma package to identify differential circRNA gene expression between MPM cell lines and normal mesothelial controls. CircRNA and microRNA interaction was predicted using Arraystar’s miRNA target prediction software based on miRanda and TargetScan algorithms.
We have identified upregulation of 290 circRNAs in MPM cell lines. Specifically, of functional importance, upregulated circRNAs derived from host genes PHKB, SLC45A4, ARHGEF28, FBXW4, TAF15, PLEKHM1, RALGPS1, STIL, L3MBTL4, ANKRD27, NHS, ILKAP, and PTK2 (mean fold change = 2.05; p<0.05) harbour predicted binding sites for tumour suppressor microRNAs miR-16, miR-15a, miR-15b, miR-34a, miR-34b, miR-34c and miR-137; that have previously been demonstrated to be downregulated in MPM tumour samples and cell lines.
CircRNAs with predicted tumour-suppressor microRNA targets were upregulated in MPM, highlighting their potential functional and diagnostic value. Validation of their expression in MPM plasma samples will be performed to test their potential as less-invasive biomarkers for MPM diagnosis.