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

描述（由申请人提供）：在接受常规筛查结肠镜检查的患者中，≥ 20%存在锯齿状结肠息肉。锯齿状息肉分为无蒂锯齿状和增生性息肉，通常很难通过内窥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，但最近的研究提供的证据表明，无蒂锯齿状息肉占所有结肠癌的20-30%。我们提出了一个多边的方法来确定分子诊断标志物的无蒂锯齿状息肉是预测增加结肠癌的风险。我们将使用最具创新性的下一代测序技术来确定20名患者血清和活检标本中完整的小RNA（< 200 nt）转录组，这些患者具有夸大的无蒂锯齿状息肉表型，称为锯齿状息肉综合征（SPS）。该综合征定义为患者在乙状结肠近端有≥ 5个无蒂锯齿状息肉，其中2个直径> 1cm，或患者在大肠任何部位有> 20个锯齿状息肉。发病年龄（通常< 40岁）和病变的多样性提示遗传易感性，但其基础尚不清楚，家族性发病也不常见。为了实现这一目标，我们开发了世界上最大的锯齿状息肉综合征患者队列之一。我们还将在20例散发性无蒂锯齿状、增生性和腺瘤性息肉患者中确定小RNA转录组，以确定一组独特的无蒂锯齿状息肉特异性microRNA（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个结肠和5个血清样本，并将在研究的第一年期间在每个队列中收集另外10个结肠和15个血清样本。基于先前的功效计算，我们确信每个队列中的20名患者将足以定义息肉类型之间的miRNA差异表达。我们将使用留一法交叉验证来开发一种两步程序，用于定义无蒂锯齿状息肉患者和传统增生性息肉患者的小RNA基因签名。我们将通过qPCR在每个队列患者的组织和血清样本中验证在无蒂锯齿状息肉患者中独特表达的10种miRNA。我们将使用mRNA 3'UTR和miRNA表达质粒的瞬时共转染、mRNA 3' UTR的定点突变和mRNA产物的蛋白质印迹来鉴定和验证选择的miRNA靶。我们的假设是，这种方法将确定分子标记物，准确区分无蒂锯齿状息肉，在锯齿状息肉综合征和散发患者，从其他息肉，并提供一个非侵入性的血清检测，以确定患者的结肠癌风险增加。其目的是对患者的结肠癌风险进行分层，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.