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

描述(申请人提供)：抗药性是癌症治疗失败的主要原因之一。转录后调控和翻译调控在化疗耐药中起着重要作用。目前，还没有可用的高通量方法来研究由少数细胞的RNA结合蛋白或非编码microRNAs介导的转录后和翻译控制。我们在拟议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)来从少量结肠癌干细胞中发现由miR-215介导的转录后调控的mRNA靶点。我们最近的研究发现miR-215参与了一些关键的抗癌靶点的调节，如胸苷合成酶和二氢叶酸还原酶。胸苷酸合酶(TS)是一种叶酸依赖性酶，催化5，10-亚甲基四氢叶酸还原甲基化生成DTMP和二氢叶酸。由于TS催化的酶反应提供了胸腺酸的唯一从头来源，胸腺酸是DNA生物合成的重要前体，TS一直是50多年来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为四氢叶酸。该反应为一碳转移反应提供了关键中间体。DHFR在叶酸动态平衡中起关键作用，是嘌呤、胸苷和某些氨基酸从头合成所必需的。因此，DHFR已成为癌症化疗的重要靶点。由miR-215转录后调控的mRNAs将通过基于多维蛋白质识别技术(MudPIT)的灵敏、高通量的蛋白质组学分析在蛋白质水平上得到验证。我们将进一步开发TRIP-SEQ方法，以提高检测罕见转录本的覆盖率和灵敏度，可能是在单细胞水平上。提出了三个具体目标：具体目标1.利用Trip-Chip方法鉴定miR-215转录后调控和翻译调控的mRNA靶点。特定目的2.通过高通量蛋白质组学分析验证miR-215介导的靶点。具体目标3.我们将通过集成下一代基于测序的表达分析(TRIP-Seq)来进一步开发Trip-Chip方法，以提高少数细胞的覆盖率和检测灵敏度。

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.