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De novo Long Noncoding RNAs and the Tumour Microenvironment in High Grade Serous Ovarian Carcinoma

高级别浆液性卵巢癌中的从头长非编码RNA和肿瘤微环境

基本信息

批准号：
1941033
负责人：
金额：
--
依托单位：
Queen Mary University of London
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1941033
关键词：
De novo Long Noncoding RNAs

项目摘要

Ovarian Cancer (OC) is the 8th most common leading cause of cancer deaths worldwide, with the highest mortality rate and worst prognosis of gynaecological cancers1. High Grade Serous Ovarian Carcinoma (HGSOC) accounts for over 70% of OC deaths. Its 5-year overall survival is less than 30% and ~80% of responders will relapse following primary treatment2. The tumour microenvironment (TME) is vital in extracellular matrix remodeling, which plays a central role in cancer initiation, progression and drug resistance3,4. Long non-protein coding RNAs (lncRNAs) are of increasing importance in tumourigenesis and prognosis and the role of lncRNAs in HGSOC and its TME is being studied5. However, the functional role of lncRNAs is still poorly understood and there remains an unmet need for novel biomarkers and therapeutic targets in HGSOC.The project aims were to;1. Establish a robust bioinformatic pipeline to identify and annotate known and novel lncRNAs.2. Obtain and annotate the top cell compartment specific (tumour vs stroma) disease-associated lncRNAs from a HGSOC discovery dataset and TCGA and ICGC validation datasets.3. Perform in-vitro validation of candidate lncRNAs.MethodsRNA-seq data was obtained from metastasised HGSOC omental biopsies (n=35). We performed read alignment and de novo transcript assembly, using the reference human genome, Gencode v29. We filtered lncRNAs using various selection criteria. This involved filtering out: mono-exonic genes, genes < 200nt in length, ensembl coding genes, ensembl pseudogenes, de novo transcripts of an unknown coding potential and ensembl genes that were not annotated as either "lincRNA", "non_coding", "antisense", "3_prime_overlapping_ncrna", "processed_transcript", "miRNA", "misc_RNA", "polymorphic_pseudogene", "processed_pseudogene" or a "pseudogene" biotype. We obtained the normalised count data and identified the disease-associated lncRNAs, as lncRNAs that significantly correlated with the HGSOC disease score6, using a Partial Least Squares Regression (PLS) model. We obtained the cell compartment specific lncRNAs using the ratio gene expression between tumour and stromal laser capture microdissected samples (n =4). Using the TCGA dataset (n=374), we validated our disease-associated and cell compartment specific lncRNAs. We also obtained lncRNAs whose expression significantly correlated with patient survival. We further validated our diseases-associated cell compartment specific lncRNAs using immune and stromal gene signatures11. Results76,322 genes, including 18,252 de novo genes aligned to the reference human genome. We identified 1523 genes as lncRNAs, including 1130 annotated and 393 de novo lncRNAs. We visualised various classes of lncRNAs including intergenic, antisense and putative enhancer de novo lncRNAs, in IGV viewer. 92 annotated and 27 novel lncRNAs significantly correlated (PLS regression cut-off) with HGSOC disease score6. Of these, 79 lncRNAs showed specificity for either tumour or stromal compartments (Log2(stroma/tumour) < |1|). Using the TCGA dataset (n =374), we validated 61 diseases-associated lncRNAs. Of these, 25 lncRNAs showed significant differences in overall survival (p< 0.05; Kaplan-Meir). Examples of these lncRNAs include DANCR, HOXB-AS3 and l_LNC141112 (a de novo lncRNA). Using Estimate's immune and stromal gene signatures11, we further validated 6 significant cell compartment specific lncRNAs (p<0.05, Student's t-test between high vs low expression of Estimate's gene signatures11). Examples of these lncRNAs include LINC00665, l_LNC14112 and MIR22HG.

卵巢癌（OC）是全球第八大最常见的癌症死亡原因，在妇科癌症中死亡率最高，预后最差1。高级别浆液性卵巢癌 (HGSOC) 占 OC 死亡人数的 70% 以上。其 5 年总生存率低于 30%，约 80% 的缓解者将在初次治疗后复发2。肿瘤微环境 (TME) 在细胞外基质重塑中至关重要，在癌症发生、进展和耐药性中发挥着核心作用3,4。长非蛋白编码 RNA (lncRNA) 在肿瘤发生和预后中变得越来越重要，并且正在研究 lncRNA 在 HGSOC 及其 TME 中的作用5。然而，lncRNA 的功能作用仍然知之甚少，HGSOC 对新型生物标志物和治疗靶点的需求仍未得到满足。该项目的目标是：1。建立强大的生物信息学管道来识别和注释已知和新型 lncRNA。2。从 HGSOC 发现数据集以及 TCGA 和 ICGC 验证数据集中获取并注释顶部细胞区室特异性（肿瘤与基质）疾病相关的 lncRNA。3。对候选 lncRNA 进行体外验证。方法RNA-seq 数据从转移的 HGSOC 大网膜活检中获得 (n=35)。我们使用参考人类基因组 Gencode v29 进行了读取比对和从头转录本组装。我们使用各种选择标准过滤lncRNA。这涉及过滤掉：单外显子基因、长度<200nt的基因、整体编码基因、整体假基因、未知编码潜力的从头转录本以及未注释为“lincRNA”、“non_coding”、“反义”、“3_prime_overlapping_ncrna”、“processed_transcript”、“miRNA”、 “misc_RNA”、“polymorphic_pseudogene”、“processed_pseudogene”或“pseudogene”生物型。我们使用偏最小二乘回归 (PLS) 模型获得了归一化计数数据，并识别了与疾病相关的 lncRNA，即与 HGSOC 疾病评分显着相关的 lncRNA。我们使用肿瘤和基质激光捕获显微切割样本之间的基因表达比率获得了细胞区室特异性 lncRNA (n = 4)。使用 TCGA 数据集 (n=374)，我们验证了与疾病相关的和细胞区室特异性的 lncRNA。我们还获得了 lncRNA，其表达与患者生存显着相关。我们使用免疫和基质基因特征进一步验证了与疾病相关的细胞区室特异性 lncRNA。结果 76,322 个基因，包括与参考人类基因组比对的 18,252 个从头基因。我们鉴定了 1523 个基因为 lncRNA，其中包括 1130 个带注释的 lncRNA 和 393 个从头 lncRNA。我们在 IGV 查看器中可视化了各种类型的 lncRNA，包括基因间、反义和推定增强子 de novo lncRNA。 92 个带注释的 lncRNA 和 27 个新型 lncRNA 与 HGSOC 疾病评分显着相关（PLS 回归截止值）6。其中，79 个 lncRNA 对肿瘤或基质区室表现出特异性 (Log2(基质/肿瘤) <|1|)。使用 TCGA 数据集 (n = 374)，我们验证了 61 种与疾病相关的 lncRNA。其中，25 个 lncRNA 在总生存期方面表现出显着差异（p < 0.05；Kaplan-Meir）。这些lncRNA的例子包括DANCR、HOXB-AS3和l_LNC141112（一种从头lncRNA）。使用 Estimate 的免疫和基质基因特征11，我们进一步验证了 6 个显着的细胞区室特异性 lncRNA（p<0.05，Estimate 基因特征的高表达与低表达之间的学生 t 检验11）。这些 lncRNA 的例子包括 LINC00665、l_LNC14112 和 MIR22HG。