Total RNA seq transcriptome profiling of thymocytes

Circular RNAs (circRNAs) are stable RNA molecules whose deregulation can drive disease and cancer mechanisms by impacting on their interactions with microRNAs and proteins and on expression of the encoded peptides. This study describes the landscape of circRNA expressed in T-cell Acute Lymphoblastic Leukemia (T-ALL). Analysis by CirComPara of RNA-seq data of 25 T-ALL patients of five genetic T-ALL subtypes and human thymocyte populations from healthy donors detected over 68,500 circRNAs. Further study of 3 447 highly expressed circRNAs derived mostly from exonic regions of 1,966 loci informed prominent differences in circRNA expression between normal and malignant T-cells, with 944 circRNAs differentially expressed in T-ALL, mostly upregulated. Next, circRNA expression signatures of different molecular genetic subgroups were defined. Examination of circRNA expression variation during thymocyte maturation and comparison with putative-cell of origin of each T-ALL subtype identified ectopic group-specific circRNAs, whose expression and backsplice sequence were confirmed in T-ALL cell lines. Our findings on circRNAs significantly extend previous data on expression of linear transcripts in subtypes of human T-ALL, opening functional investigation of deregulated circRNAs so far uncharacterized in human T-ALL, including a few for which oncogenic roles or molecular functions have been already proven in different settings. Profiling of the total RNA transcriptome of different CD34+ and DP stages of T cell development isolated from human postnatal thymus, for two or three donors.环状RNA（circRNAs）是一类稳定的RNA分子，其表达失调可通过影响与微小RNA（microRNAs）、蛋白质的相互作用，以及调控编码肽的表达，参与疾病与癌症的发生发展机制。本研究刻画了T细胞急性淋巴细胞白血病（T-cell Acute Lymphoblastic Leukemia, T-ALL）中的环状RNA表达谱。本研究借助CirComPara工具，对来自5种遗传亚型T-ALL的25例患者，以及健康供体的人胸腺细胞群体的RNA测序（RNA-seq）数据开展分析，共检测到超过68500种环状RNA。针对主要源自1966个基因座（loci）外显子区域（exonic regions）的3447种高表达环状RNA的进一步研究，揭示了正常T细胞与恶性T细胞之间环状RNA表达的显著差异：在T-ALL样本中共存在944种差异表达的环状RNA，其中多数呈现上调趋势。随后，本研究明确了不同分子遗传亚型的环状RNA表达特征。通过检测胸腺细胞成熟过程中环状RNA的表达变化，并与每种T-ALL亚型的推定起源细胞进行比对，本研究鉴定出了亚型特异性的异位表达环状RNA，其表达特征与反向剪接序列（backsplice sequence）均在T-ALL细胞系中得到验证。本研究关于环状RNA的发现，显著拓展了此前针对人类T-ALL亚型线性转录本（linear transcripts）表达的相关数据，为目前尚未在人类T-ALL中被表征的失调环状RNA的功能研究提供了方向——其中部分环状RNA的致癌作用或分子功能已在其他研究场景中得到证实。本研究同时对2至3名健康供体、分离自人类出生后胸腺的不同发育阶段（CD34阳性（CD34+）及双阳性（DP）阶段）的T细胞总RNA转录组开展了表达谱分析。