oa The Role of circRNA on EMT Induced Ovarian Cancer Cells

Circular RNA (circRNA) represent a large class of noncoding RNAs that were previously considered as possible artifacts of abnormal RNA splicing, however, recent studies has shown that circRNA play an important role in regulating gene expression in mammals. Unlike linear RNAs, the downstream 5’ (splice donor) and upstream 3’ (splice acceptor) join together to form a closed continuous loop and this is one of the ways circRNA are formed. These exons are referred to as scrambled exons or shuffled exons and the process is known as backsplicing. circRNA can also be formed from lariat introns which escape debranching. Epithelial - mesenchymal transition (EMT) is a reversible process in which cells loses their epithelial phenotype and obtain mesenchymal phenotype. Moreover, EMT decreases expression of epithelial marker genes such as E-cadherin and increases expression of mesenchymal marker genes such as Mucin, N-cadherin, MMP2, Snail, Twist, VIM, FN, ITGA, FOX, TGFβ. Thus, EMT can play a major role in facilitating the migration, invasion and progression of cancerous cells in human body tissue. Several studies showed the regulatory role of linear forms of RNA transcripts (mRNA) on EMT, this study aims to highlight the regulatory role of circRNA in EMT.

Two epithelial ovarian cancer cell lines (CaOV3 and SKOV3) were treated with EMT inducing media supplement. RNA was isolated using all prep DNA/RNA kit. iScript cDNA synthesis kit was used for cDNA synthesis and reverse transcription. SYBR select master mix kit was used for PCR amplification. Product size was checked on 2.2% agarose gel (flash gel DNA cassettes-Lonza). CircRNAs with clear prominent bands were selected for gene expression analysis using eleven EMT signature genes by real time PCR. Migration scratch assay was used to test the ability of cells to migrate when subjected to EMT inducing media. Cells were seeded in a 6 well plate, and when they reached 80% confluency, a scratch was made using 1 ml tip. EMT media was added, and cells were kept in serum free media for 12 hours interval. Distance migrated by cells was measured in both treated and untreated wells using a Zeiss microscope. Extracted RNA from treated and untreated cells with commercially available kits was prepared for Illumina paired-end sequencing. Illumina deep sequencing using HiSeq 2500 yielded an average of 30 million read pairs per library of 100 bp read length. Using an in-house developed computational pipeline we identified and characterized the circRNA expression in EMT versus non-induced cells and compared it with the linear (mRNA) expression.

Real time PCR assay with eleven EMT signature genes showed a complete epithelial to mesenchymal transition after EMT supplement media was induced in both cell ovarian cancer cell lines (CaOV3 and SKOV3). Scratch assay showed cells which were subjected to EMT media migrated to the middle of the well and almost closed the scratch gap. However, cells in untreated wells neither showed motility nor migration. RNA-sequencing data showed that a large number of candidate circRNAs and mRNAs are differentially expressed between Epithelial and Mesenchymal states and belong to well characterized EMT markers like E-cadherin, N-Cadherin, Fibronectin, Snail and Vimentin genes. We further report that compared to mRNAs, circRNAs show a stronger differential expression trend for EMT related biochemical pathways like tight junctions,gap junctions and adherens junctions indicating a regulatory role for circRNAs in Epithelial to Mesenchymal transition.

In conclusion, our results clearly demonstrates the potential role circRNA has on EMT induced cancer cells. In future, further analysis will be done to investigate the regulatory potential of the circRNA forms by using knockdown based assay.