High-throughput sequencing identifies 25,303 circRNA targets, including 20,036 known circRNAs and 5,267 uncharacterized circRNAs, in gastric cancer tissues

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. Overall design: Three pairs of gastric cancer tissues and their corresponding adjacent non-cancerous tissues were detected by circRNA-sequencing.

研究目的：随着人类基因组测序计划的推进，环状RNA（circRNAs）因其稳定的环状结构，在癌症研究领域受到广泛关注。本研究旨在鉴定胃癌（Gastric Cancer, GC）组织中差异表达的环状RNA，并探讨其在胃癌诊断与治疗中的潜在作用。
研究方法：采用德国Magen公司的Hipure Total RNA Mini Kit提取组织总RNA。使用美国加利福尼亚州卡尔斯巴德市Invitrogen公司的Qubit 3.0荧光分光光度计检测RNA浓度，采用美国加利福尼亚州卡尔斯巴德市Applied Biosystems公司的Agilent 2100生物分析仪评估RNA完整性，RIN值≥7.0的样本视为合格。取约2μg总RNA，使用适配Illumina®测序平台的KAPA RNA HyperPrep Kit with RiboErase (HMR)建库试剂盒（Kapa Biosystems公司，美国马萨诸塞州沃本市）构建RNA测序文库。简要流程如下：去除核糖体RNA后，将总RNA与10单位RNase R（Epicentre Technologies公司，美国麦迪逊市）于37℃孵育30分钟；随后对经RNase R处理的去核糖体RNA进行片段化，依次完成第一链cDNA合成及定向第二链cDNA合成；之后对纯化后的cDNA进行A尾加尾及接头连接，最终扩增得到纯化的带接头连接的DNA片段。将每个文库稀释至10 nM，按等摩尔浓度混合后进行簇生成。所有样本均采用双端（PE150）测序模式进行测序。
研究结果：本研究共检测并注释得到25303个环状RNA靶点，其中包含20036个已知环状RNA及5267个未注释的新型环状RNA。在胃癌组织中，共鉴定出2007个表达存在显著统计学差异的环状RNA，筛选标准为差异倍数>2.0且P<0.05。
实验设计：选取3对胃癌组织及其对应的癌旁正常组织，通过circRNA测序进行检测分析。