Molecular mechanisms for regulating microRNA levels in metazoans

调节后生动物 microRNA 水平的分子机制

• 批准号: 10622724
• 负责人: MINGYI XIE
• 金额: $ 41.18万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-13 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622724
• 关键词: 3' Untranslated Regions; Address; Affect; Base Pairing; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Cell Line; Colorectal Cancer; Control Animal; Cultured Cells; DNA; Defect; Degradation Pathway; Development; Disease; Doxycycline; Engineering; Enzymes; Essential Genes; Gene Expression; HCT116 Cells; In Vitro; Individual; Knowledge; Malignant Neoplasms; Messenger RNA; MicroRNAs; Molecular; Molecular Probes; Neurodegenerative Disorders; Pathologic; Process; Property; Proteins; RNA; Regulation; Research; Small RNA; Structure; Techniques; Therapeutic; Tissues; Transcript; Untranslated RNA; bioinformatics pipeline; design; experience; genetic information; improved; model organism; novel therapeutics; tool; tumorigenesis

PROJECT SUMMARY Gene expression, the genetic information flow from DNA to RNA to protein, involves delicate regulation by a group of small non-coding RNAs called microRNAs (miRNAs). Dysregulated miRNA networks have been observed in a variety of disease states including cancer, developmental defects, and neurodegenerative diseases. In these disease states, the miRNA abundance can be affected at either the degradation or the biogenesis steps. Target-directed miRNA degradation (TDMD) is a mechanism by which specific miRNAs undergo degradation when extensively base-paired with their target RNAs. How TDMD may contribute to different diseases is unclear due to the paucity of known target RNAs (or triggers) that can induce miRNA degradation. On the other hand, Dicer functions as the central enzyme in processing precursor miRNAs into mature miRNAs. Dicer deficiency promotes tumorigenesis, but how different miRNAs are affected by lower levels of Dicer in cancer remains a question. To address these knowledge gaps, we will probe the molecular regulatory mechanisms of specific miRNA abundance in metazoans focusing on three projects: 1. Expand the repertoire of metazoan TDMD triggers. We have established CLASH, a combined biochemical and bioinformatics pipeline, for identification of TDMD triggers in cultured cells. We will further improve CLASH for application in model organisms to identify conserved and essential TDMD triggers. 2. Investigate the properties of effective TDMD triggers. Known TDMD trigger sequences are concentrated in conserved regions of the mRNA 3′ UTR and non-coding RNAs. We will investigate the importance of trigger transcripts sequences, structure and the non-coding properties in inducing TDMD. 3. Differential Dicer processing of cellular miRNAs. In an engineered colorectal cancer HCT116 cell line, in which Dicer expression can be fine-tuned by doxycycline, we will determine the miRNA profile at different Dicer expression levels. Determinants for differential responsiveness to Dicer of individual miRNAs will be probed by in vitro Dicer cleavage assays and identification of miRNA binding proteins. Our unique experimental approaches will provide the basis for developing novel therapeutics through modulation of the TDMD pathway and Dicer action. Techniques and experience acquired in this project will prepare our group for future research in exploring molecular machineries that regulate small RNAs essential for gene expression in normal and diseased tissue.

项目摘要 基因表达，即遗传信息从DNA到RNA再到蛋白质的流动，涉及到一个基因的精细调控。 一组小的非编码RNA称为microRNA（miRNA）。失调的miRNA网络已经被 在多种疾病状态中观察到，包括癌症、发育缺陷和神经退行性疾病。 疾病在这些疾病状态下，miRNA丰度可以在降解或降解过程中受到影响。 生物发生步骤靶向miRNA降解（TDMD）是一种特异性miRNA 当与它们的靶RNA进行广泛的碱基配对时会发生降解。TDMD如何有助于 由于缺乏可以诱导miRNA的已知靶RNA（或触发因子）， 降解另一方面，Dicer在将前体miRNAs加工成 成熟的miRNAs。Dicer缺乏促进肿瘤发生，但不同的miRNAs如何受到较低的 癌症中Dicer的水平仍然是一个问题。为了解决这些知识缺口，我们将探索分子 后生动物中特异性miRNA丰度的调控机制集中在三个项目： 1.扩展后生动物TDMD触发器的所有功能。 我们已经建立了CLASH，一个生物化学和生物信息学相结合的管道，用于识别TDMD 在培养的细胞中触发。我们将进一步改进CLASH用于模式生物的识别， 保守的和必需的TDMD触发物。 2.研究有效TDMD触发器的属性。 已知的TDMD触发序列集中在mRNA 3′ UTR的保守区域和非编码区， RNA。我们将研究触发转录物序列、结构和非编码区的重要性， 诱导TDMD的特性。 3.细胞miRNA的差异切酶加工。 在工程化的结肠直肠癌HCT116细胞系中，其中Dicer表达可以通过 在多西环素的作用下，我们将确定不同Dicer表达水平下的miRNA谱。的决定因素 将通过体外Dicer切割测定来探测单个miRNA对Dicer的不同响应性， miRNA结合蛋白的鉴定。 我们独特的实验方法将为开发新的治疗方法提供基础， 调节TDMD途径和Dicer作用。在这个项目中获得的技术和经验将 准备我们的小组为未来的研究，探索分子机制，调节小RNA的基本 基因在正常和病变组织中的表达。

项目成果

Towards an integrative understanding of cancer mechanobiology: calcium, YAP, and microRNA under biophysical forces

全面了解癌症力学生物学：生物物理力下的钙、YAP 和 microRNA

• DOI: 10.1039/d1sm01618k
• 发表时间: 2022
• 期刊: Soft Matter
• 影响因子: 3.4
• 作者: Liang, Chenyu;Huang, Miao;Li, Tianqi;Li, Lu;Sussman, Hayley;Dai, Yao;Siemann, Dietmar W.;Xie, Mingyi;Tang, Xin
• 通讯作者: Tang, Xin

Fluorescent In Situ Detection of RNA-Protein Interactions in Intact Cells by RNA-PLA.

通过 RNA-PLA 荧光原位检测完整细胞中的 RNA-蛋白质相互作用。

• DOI: 10.1007/978-1-0716-3191-1_13
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Li,Tianqi;Zhang,Wei;Xie,Mingyi
• 通讯作者: Xie,Mingyi

YAP at the Crossroads of Biomechanics and Drug Resistance in Human Cancer.

• DOI: 10.3390/ijms241512491
• 发表时间: 2023-08-06
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Huang, Miao;Wang, Heyang;Mackey, Cole;Chung, Michael C. C.;Guan, Juan;Zheng, Guangrong;Roy, Arkaprava;Xie, Mingyi;Vulpe, Christopher;Tang, Xin
• 通讯作者: Tang, Xin

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

通过荧光显微镜和分子工具对体外和离体人类癌细胞中的 Ca2 信号进行功能询问。

• DOI: 10.1007/978-1-0716-3271-0_7
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Liang,Chenyu;Huang,Miao;Tanaka,Mai;Lightsey,Suzanne;Temples,Madison;Lepler,SharonE;Sheng,Peike;Mann,WilliamP;Widener,AdrienneE;Siemann,DietmarW;Sharma,Blanka;Xie,Mingyi;Dai,Yao;Phelps,Edward;Zeng,Bo;Tang,Xin
• 通讯作者: Tang,Xin

Biological features between miRNAs and their targets are unveiled from deep learning models.

• DOI: 10.1038/s41598-021-03215-w
• 发表时间: 2021-12-10
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Gu T;Xie M;Barbazuk WB;Lee JH
• 通讯作者: Lee JH