Loss of function shRNA screen on circRNA and linear genes in PCa cell lines. Loss of function shRNA screen on circRNA and linear genes in PCa cell lines

Standard RNA analyses using microarrays and low-coverage polyadenylation enriched RNA-Sequencing (RNA-Seq) cannot fully characterize the complexity of the cancer transcriptome. To fully elucidate the transcriptome of prostate tumours, we performed ultra-deep total RNA-Seq on 144 localized prostate tumours with long-term clinical follow up. Analysis of linear RNAs identified a transcriptomic subtype associated with the aggressive intraductal carcinoma subhistology, and a fusion gene profile that differentiates localized from metastatic prostate cancers. Analysis of back-splicing events identified widespread RNA circularization, with the average tumour expressing 7,140 distinct circular RNAs. The degree of aberrant circRNA production is correlated to disease progression in multiple clinical cohorts. Loss of function screens identified 11.3% of the screened circRNAs as essential to prostate cancer proliferation, and for 93.6% of these, their parental linear genes are not required for proliferation. Follow-up studies on circCSNK1G3 revealed its role in regulating cell cycle progression. Ultra-deep transcriptome sequencing thus provides a more comprehensive view of the linear and circular transcriptional and functional landscapes of localized prostate cancer. Overall design: Functional genomic screen with shRNA on circRNA and linear genes was carried out in 4 PCa cell lines (LNCaP, 22Rv1, PC-3 and V16A).

传统基于微阵列（microarrays）与低覆盖度聚腺苷酸化富集RNA测序（RNA-Seq）的标准RNA分析，无法完全解析癌症转录组的复杂特征。为全面阐明前列腺肿瘤的转录组全貌，我们对144例具备长期临床随访数据的局限性前列腺肿瘤开展了超深度总RNA测序。对线性RNA的分析鉴定出与侵袭性导管内癌亚组织学类型相关的转录组亚型，以及可区分局限性前列腺癌与转移性前列腺癌的融合基因谱。对反向剪接事件的分析揭示了广泛存在的RNA环化现象：每例肿瘤平均表达7140种不同的环状RNA（circular RNAs）。异常环状RNA生成的程度与多个人类临床队列中的疾病进展显著相关。功能缺失筛选实验显示，11.3%的受试环状RNA为前列腺癌细胞增殖所必需，且其中93.6%的环状RNA对应的亲本线性基因并非细胞增殖所必需。针对circCSNK1G3的后续研究揭示了其在调控细胞周期进程中的功能。因此，超深度转录组测序可更为全面地展现局限性前列腺癌的线性与环状转录组及功能图谱。
实验整体设计：针对环状RNA与线性基因开展的短发夹RNA（shRNA）功能基因组筛选，在4株前列腺癌细胞系（PCa）——LNCaP、22Rv1、PC-3及V16A中完成。