ABSTRACT: Purpose: With the advent of human genome sequencing project, circRNAs attracted widespread attention in cancer research due to its stable ring structure. Our aim was to identify differentially expressed circRNAs in GC and explore their potential roles in GC diagnosis and treatment. Methods: Total RNA from tissues was isolated by Hipure Total RNA Mini Kit (Magen, Germany). Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, California) was used for RNA concentration measurement while Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA) for RNA integrity estimation. A RIN value over 7.0 was considered eligible. RNA-seq library was prepared with approximately 2μg of total RNA using KAPA RNA HyperPrep Kit with RiboErase (HMR) for Illumina® (Kapa Biosystems, Inc., Woburn, MA). Briefly, total RNA was incubated at 37 °C for 30 min with 10 units RNase R (Epicentre Technologies, Madison, USA) after removing ribosomal RNA. Next, the ribominus RNase R (+) RNAs was fragmented and then first strand and directional second strand synthesis were performed. Then the A tailing and adapter ligation were performed with the purified cDNA. Finally, the purified, adapter-ligated DNA was amplified. Each library was diluted to 10 nM and pooled equimolar prior to clustering. Paired-End (PE150) sequencing was performed on all samples. Results: A total of 25,303 circRNA targets, including 20,036 known circRNAs and 5,267 uncharacterized circRNAs, were detected and defined. Among them, there were 2,007 circRNAs with statistically significant differences in expression in GC tissues, in which fold changes>2.0 and P<0.05 were identified.
