Annotation and functional characterization of long noncoding RNAs deregulated in pancreatic adenocarcinoma. Annotation and functional characterization of long noncoding RNAs deregulated in pancreatic adenocarcinoma

Purpose: Transcriptome analysis of pancreatic ductal adenocarcinoma (PDAC) has been useful to identify gene expression changes that sustain malignant phenotypes. Yet, most studies examined only tumor tissues and focused on protein-coding genes, leaving long non-coding RNAs (lncRNAs) largely underexplored. Methods: We generated total RNA-Seq data from patient-matched tumor and nonmalignant pancreatic tissues and implemented a computational pipeline to survey known and novel lncRNAs. siRNA-mediated knockdown in tumor cell lines was performed to assess the contribution of PDAC-associated lncRNAs to malignant phenotypes. Gene co-expression network and functional enrichment analyses were used to assign deregulated lncRNAs to biological processes and molecular pathways. Results: We detected 9,032 GENCODE lncRNAs as well as 523 unannotated lncRNAs, including transcripts significantly associated with patient outcome. Aberrant expression of a subset of novel and known lncRNAs was confirmed in patient samples and cell lines. siRNA-mediated knockdown of a subset of these lncRNAs (LINC01559, LINC01133, CCAT1, LINC00920 and UCA1) reduced cell proliferation, migration and invasion. Gene co-expression network analysis associated PDAC-deregulated lncRNAs with diverse biological processes, such as cell adhesion, protein glycosylation and DNA repair. Furthermore, UCA1 knockdown was shown to specifically deregulate co-expressed genes involved in DNA repair and to negatively impact DNA repair following damage induced by ionizing radiation. Conclusions: Our study expands the repertoire of lncRNAs deregulated in PDAC, thereby revealing novel candidate biomarkers for patient risk stratification. It also provides a roadmap for functional assays aimed to characterize novel mechanisms of action of lncRNAs in pancreatic cancer, which could be explored for therapeutic development. Overall design: Strand-oriented total RNA-seq libraries from 15 paired samples (tumor and non-tumor adjacent pancreatic tissue) were sequenced using Illumina HiSeq 1500/2500 platform

研究目的：胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）的转录组分析，可用于识别维持恶性表型的基因表达改变。然而，绝大多数现有研究仅针对肿瘤组织展开，且聚焦于蛋白编码基因，导致长链非编码RNA（long non-coding RNAs, lncRNAs）的相关研究严重不足。
研究方法：本研究从患者配对的肿瘤与非恶性胰腺组织中获取样本，生成总RNA测序（total RNA-Seq）数据，并搭建生物信息学分析流程，以系统调研已知及新型lncRNAs。我们在肿瘤细胞系中开展小干扰RNA（siRNA）介导的基因敲低实验，以评估PDAC相关lncRNAs对恶性表型的调控作用。此外，通过基因共表达网络分析与功能富集分析，将失调的lncRNAs关联至特定生物学过程与分子通路。
研究结果：本研究共检测到9032个GENCODE注释的lncRNAs，以及523个未注释的lncRNAs，其中部分转录本与患者预后显著相关。我们在患者样本与细胞系中验证了部分新型及已知lncRNAs的异常表达。对其中5个lncRNAs（LINC01559、LINC01133、CCAT1、LINC00920及UCA1）进行siRNA介导的敲低后，肿瘤细胞的增殖、迁移与侵袭能力均受到显著抑制。基因共表达网络分析显示，PDAC失调的lncRNAs与多种生物学过程密切相关，包括细胞黏附、蛋白质糖基化及DNA修复。进一步实验表明，敲低UCA1可特异性调控参与DNA修复的共表达基因，并对电离辐射诱导损伤后的DNA修复过程产生负面影响。
研究结论：本研究拓展了PDAC中失调lncRNAs的已知种类，为患者风险分层提供了新型候选生物标志物。同时，本研究为后续功能实验提供了研究框架，以解析lncRNAs在胰腺癌中的新型作用机制，相关机制可进一步探索用于治疗开发。
整体实验设计：本研究针对15对配对样本（胰腺肿瘤组织与癌旁非肿瘤组织）构建链特异性总RNA测序文库，并使用Illumina HiSeq 1500/2500平台完成测序。