Non-coding RNAs in serrated polyps identify patients with high colon cancer risk

描述（由申请人提供）：在接受常规结肠镜筛查的患者中，有20%的患者出现锯齿状结肠息肉。锯齿状息肉分为无梗锯齿状息肉和增生性息肉，通常难以通过内镜和组织学检查进行区分。虽然锯齿状息肉以前被认为是无害的，但最近的研究提供证据表明，无柄锯齿状息肉占所有结肠癌的20-30%。我们提出了一种多边方法来确定可预测结肠癌风险增加的无柄锯齿状息肉的分子诊断标记。我们将使用最具创新性的下一代测序技术来确定20例无根锯齿状息肉（即锯齿状息肉综合征，SPS）患者的血清和活检标本中完整的小RNA （< 200nt）转录组。该综合征定义为：在乙状结肠近端出现= 5个无梗锯齿状息肉，其中2个直径为> ~ 1cm，或在大肠任何部位出现> ~ 20cm锯齿状息肉。表现的年龄（通常小于40岁）和病变的多样性提示遗传易感性，但其基础尚不清楚，家族性发生并不常见。为了实现这一目标，我们开发了世界上最大的锯齿状息肉病综合征患者队列之一。我们还将定义20例散发性无柄锯齿状息肉、增殖性息肉和腺瘤性息肉患者的小RNA转录组，以鉴定一组独特的无柄锯齿状息肉特异性microRNAs （miRNAs）。小rna的分析将有助于区分息肉类型，类似于先前发表的使用miRNA面板的肿瘤分类研究。我们已经在每个队列中收集了10-20份结肠和5份血清样本，并将在研究的第一年在每个队列中再收集10份结肠和15份血清样本。根据先前的功率计算，我们确信每个队列中20例患者足以确定不同息肉类型的mirna差异表达。我们将使用留一交叉验证来开发一个两步程序，用于确定无柄锯齿状息肉患者和传统增长性息肉患者的小RNA基因特征。我们将通过qPCR在每个队列患者的组织和血清样本中验证10个在无柄锯齿状息肉患者中唯一表达的mirna。我们将利用mRNA 3' UTRs和miRNA表达质粒的瞬时共转染、mRNA 3' UTRs的定点诱变和mRNA产物的western blotting来鉴定和验证选择的miRNA靶点。我们的假设是，这种方法将识别分子标记，准确区分无柄锯齿状息肉综合征和零星患者的无柄锯齿状息肉与其他息肉，并为识别结肠癌风险增加的患者提供无创血清检测。目的是对患者患结肠癌的风险进行分层，以指导癌症筛查试验的频率，并帮助设计化学预防试验。

DESCRIPTION (provided by applicant): Serrated colon polyps are present in = 20% of patients undergoing routine screening colonoscopy. Serrated polyps are divided into sessile serrated and hyperplastic polyps and are often difficult to differentiate by endoscopic and histological examination. Although serrated polyps were previously considered harmless, recent studies provide evidence that sessile serrated polyps account for 20-30% of all colon cancers. We propose a multilateral approach to identify molecular diagnostic markers for sessile serrated polyps that are predictive of increased colon cancer risk. We will use the most innovative next generation sequencing technology to define the complete small RNA (< 200 nt) transcriptome in both serum and biopsy specimens from 20 patients with an exaggerated phenotype of sessile serrated polyps, known as serrated polyposis syndrome (SPS). The syndrome is defined as patients with = 5 sessile serrated polyps proximal to the sigmoid colon, two of which are > 1cm diameter, or patients with > 20 serrated polyps at any site in the large bowel. The age of presentation (often < 40 yrs) and multiplicity of lesions suggests a genetic predisposition, but it basis remains unknown and familial occurrence is unusual. To achieve this goal we have developed one of the largest cohorts of patients with the serrated polyposis syndrome in the world. We will also define the small RNA transcriptome in 20 patients with sporadic sessile serrated, hyperplastic and adenomatous polyps to identify a unique panel of microRNAs (miRNAs) specific to sessile serrated polyps. The analysis of small RNAs will aid in differentiating between polyp types similar to previously published tumor classification studies using miRNA panels. We have already collected 10-20 colon and 5 serum samples in each cohort and will collect another 10 colon and 15 serum samples in each cohort during the first year of study. Based on previous power calculations we are confident 20 patients in each cohort will be adequate to define differential expression of miRNAs across polyp types. We will use leave-one-out cross-validation to develop a two-step procedure for defining small RNA gene signatures in patients with sessile serrated polyps and patients with traditional hyperplastic polyps. We will validate ten miRNAs uniquely expressed in patients with sessile serrated polyps by qPCR in tissue and serum samples from patients in each cohort. We will identify and validate select miRNA targets using transient co-transfection of mRNA 3' UTRs and miRNA expression plasmids, site-directed mutagenesis of mRNA 3' UTRs and western blotting of mRNA products. Our hypothesis is that this approach will identify molecular markers that accurately differentiate sessile serrated polyps, in both the serrated polyposis syndrome and sporadic patients, from other polyps and provide a non-invasive serum test for identifying patients with an increased risk for colon cancer. The objective is to stratify patient's risk for colon cancer to guide the frequeny of cancer screening tests and aid the design of chemoprevention trials.