Identification of post-transcriptionally regulated targets by TrIP-Chip/Seq

描述（申请人提供）：化疗耐药是癌症治疗失败的主要原因之一。转录后和翻译控制在化学耐药中起重要作用。目前还没有高通量的方法来研究由RNA结合蛋白或来自少数细胞的非编码microrna介导的转录后和翻译控制。我们在该项目中的主要目标是应用我们新开发的翻译免疫沉淀-微阵列分析（TrIP-Chip）(1)从少数结肠癌干细胞中发现miR-215介导的转录后调控mRNA靶点。我们最近的研究表明，miR-215参与调节一些关键的抗癌靶点，如胸苷酸合成酶和二氢叶酸还原酶。胸苷酸合成酶（TS）是一种叶酸依赖性酶，它催化5,10-亚甲基四氢叶酸对dUMP的还原甲基化，形成dTMP和二氢叶酸。由于TS催化的酶促反应提供了细胞内胸腺苷酸（DNA生物合成的必要前体）的唯一从头来源，因此TS在50多年来一直是癌症化疗的重要靶点。二氢叶酸还原酶（DHFR）催化nadph依赖的二氢叶酸还原成四氢叶酸。该反应是单碳转移反应的关键中间体。DHFR在叶酸稳态中起关键作用，是嘌呤、胸苷酸和某些氨基酸从头合成所必需的。因此，DHFR已成为癌症化疗的关键靶点。miR-215转录后调控的mrna将通过基于多维蛋白鉴定技术（MudPIT）的敏感、高通量鸟枪式蛋白质组学分析在蛋白水平上进行验证。我们将进一步开发TrIP-Seq方法，以增加在单细胞水平上检测罕见转录本的覆盖率和灵敏度。提出了三个具体目标：使用TrIP-Chip方法鉴定miR-215转录后和翻译调控的mRNA靶点。具体目标2。通过高通量蛋白质组学分析验证miR-215介导的靶标。具体目标3。我们将通过整合下一代基于测序的表达分析（TrIP-Seq）进一步开发TrIP-Chip方法，以增加对少数细胞的覆盖范围和检测灵敏度。

DESCRIPTION (provided by applicant): Resistance to chemotherapy is one of the major reasons for the failure of cancer treatment. Post- transcriptional and translational control plays an important role in chemoresistance. Currently there is no available high throughput approach to investigate post-transcriptional and translational control mediated by RNA binding proteins or non-coding microRNAs from a small number of cells. Our primary objective in the proposed project is to apply our newly developed Translational Immunoprecipitation-Microarray Analysis (TrIP-Chip) (1) to discover post-transcriptionally regulated mRNA targets mediated by miR-215 from a small number of colon cancer stem cells. Our recent studies revealed that miR-215 is involved in regulating some key anticancer targets, e.g. thymidylate synthase and dihydrofolate reductase. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to form dTMP and dihydrofolate. Because the TS-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis, TS has been an important target for cancer chemotherapy for over 50 years. The enzyme dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction provides the key intermediate in one-carbon transfer reactions. DHFR plays a critical role in folate homeostasis, and is required for the de novo synthesis of purines, thymidylate and certain amino acids. Therefore, DHFR has served as a critical target in cancer chemotherapy. The mRNAs regulated post-transcriptionally by miR-215 will be validated at the protein level by a sensitive, high throughput shotgun proteomic analysis based on multi-dimensional protein identification technology (MudPIT). We will further develop a TrIP-Seq approach to increase both the coverage and sensitivity to detecting rare transcripts potentially at the single cell level. Three specific aims are proposed: Specific Aim 1. Identify post-transcriptionally and translationally regulated mRNA targets of miR-215 using TrIP-Chip approach. Specific Aim 2. Validate miR-215 mediated targets by high throughput proteomic analysis. Specific Aim 3. We will further develop the TrIP-Chip approach by integrating next generation sequencing based expression analysis (TrIP-Seq) to increase coverage and detection sensitivity from a small number of cells.