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Arsenic-Induced miRNA-199 and mriRNA-214 Deplete Mitochondrial DNA for the Generation of Cancer Stem-Like Cells

砷诱导的 miRNA-199 和 mRNA-214 消耗线粒体 DNA 以生成癌症干细胞样细胞

基本信息

批准号：
10489836
负责人：
Fei Chen
金额：
$ 27.15万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10489836
关键词：
Area Arsenic Binding Biochemical Bladder CRISPR/Cas technology Cancer Model Carcinogens Case-Control Studies Cell Line Cells ChIP-seq Characteristics Chromatin Chromatin Structure Clinical DNA Methylation DNA biosynthesis Data Deposition Dose Environmental Exposure Epigenetic Process Epithelial Cells Exhibits Exposure to Gene Expression Profile Generations Genes Genetic Genetic Transcription Geology Glucose Glycolysis Goals Histone H3 Human Impairment In Vitro International Agency for Research on Cancer Lead Link Liver Lung Lysine MAPK8 gene Malignant - descriptor Malignant Neoplasms Malignant neoplasm of lung Maps Metabolic Metabolic Pathway Metabolism Metals MicroRNAs Mitochondria Mitochondrial DNA Molecular Monitor Mus NOD/SCID mouse Non-Small-Cell Lung Carcinoma Organ Oxidative Phosphorylation Pathway interactions Phosphorylation Planet Earth Preclinical Testing Production Prostate Public Health Reporting Research Research Project Grants Risk Role S-Adenosylhomocysteine STAT3 gene Serine Signal Transduction Skin Testing Transcriptional Regulation base bronchial epithelium c-myc Genes cancer cell cancer stem cell cancer therapy carcinogenesis carcinogenicity cell transformation drinking water embryonic stem cell exposed human population ground water histone methylation in vivo knock-down metabolomics mitochondrial dysfunction mitochondrial metabolism mouse model mtTF1 transcription factor notch protein novel therapeutic intervention overexpression pluripotency population based promoter response self-renewal stem-like cell stemness targeted treatment transcription factor transcriptome sequencing transcriptomics tumor

项目摘要

We have shown that consecutive treatment of the human bronchial epithelial cells with the environmentally relevant concentration of As3+ (0.125 – 0.25M), an environmental metalloid metal, for six months, induces transformation of the human bronchial epithelial cells, some of which possess characteristics of the cancer stem-like cells (CSCs), such as tumor sphere formation in vitro, self- renewal in vivo, increased expression of the stemness genes, including Oct4, Sox2, KLF4, and c-myc. In addition, these cancer stem-like cells exhibited a pronounced increase in the expression of several microRNAs, most notably, the miR-214, miR-199, miR-10b, miR-34b, etc. Furthermore, integrated transcriptomic and metabolomic analyses demonstrated a higher rate of glycolysis and lower levels of mitochondrial metabolism due to mitochondrial DNA (mtDNA) depletion among these As3+-induced CSCs. Lastly, a unique glycolytic feature that is different from naïve embryonic stem cells (ESCs) and cancer cells was found in these As3+-induced CSCs. Both ESCs and cancer cells direct glycolysis for lactate production. In contrast, the As3+-induced CSCs show increased conversion of the glycolytic intermediates into the subsidiary pathways for the generation of N-acetylglucosamine important for O- GlcNAcylation of the stemness genes and the S-adenosyl methionine (SAM) that contributes to DNA and histone methylation. Accordingly, the goal of this application is to determine: (1) is As3+-induced miRNAs, esp. miR214/199, responsible for the depletion of mtDNA and the consequent inhibition of mitochondria; (2) if so, how miRNAs induced by As3+ impairs the integrity and function of mtDNA and mitochondria; and (3) how the impaired function of mitochondria contributes to the generation of the CSCs induced by As3+. We hypothesize that As3+-induced JNK-dependent pSTAT3S727 and miR-214/199 switch mitochondrial OXPHOS to glycolysis for the formation of CSCs. To test this hypothesis, the following three specific aims are proposed: Specific Aim 1: As3+-activated JNK and pSTAT3S727 enforce expression of miR-214 and miR-199 that down-regulate mitochondrial transcription factor A (TFAM) in BEAS-2B and other lung cells for the generation of CSCs. We will focus on the transcriptional regulation of the miR-214/199 cluster with emphases on promoter DNA methylation and transcription factor binding in cellular response to As3+ and its down-stream signaling; Specific aim 2: Understand how As3+-induced JNK, miR-214/199 and mitochondrial dysfunction contribute to the formation of CSCs with an emphasis on metabolic reprogramming from OXPHOS to glycolysis. Specific Aim 3: Defining the causal roles of As3+-induced JNK- and miR-214/199-dependent metabolic reprogramming in the changes of epigenetics related to chromatin structure and accessibility that linked to self-renewal and/or differentiation of the As3+-induced CSCs through high-throughput profiling. We will identify metabolite- dependent epigenetic and chromatin changes in non-transformed cells, As3+-induced transformed cells and CSCs, which will be further verified through overexpressing or CRISPR-Cas9-based knockdown of the key genes in the related metabolic pathways and monitoring the self-renewal and differentiation status of the CSCs. The high-throughput approaches will include ChIP-seq to map H3K9me3, H3K27me3, and H3K4me3, and RNA-seq to profile transcription of the genes, esp. for those contributing to the pluripotency, self-renewal and differentiation of the CSCs. We anticipate that the results from the proposed studies will unravel importance of As3+-induced miR-214/199 on the generation of CSCs and lead to emerging of new concepts of As3+ carcinogenesis by emphasizing the capability of As3+ in CSC induction. Moreover, we believe that the date generated from this project will help us in developing novel therapeutic strategies by targeting JNK, miR-214/199 and CSCs through utilizing our unique mouse orthotopical lung cancer model in NOD/SCID mice in a separate research project.

我们已经表明，用人支气管上皮细胞连续处理，环境相关浓度的As 3+（0.125 - 0.25 μ M），一种环境类金属，六个月，诱导人支气管上皮细胞的转化，其中一些细胞具有癌症干细胞样细胞（CSCs）的特征，如体外肿瘤球形成、自身体内更新，增加了干性基因的表达，包括Oct 4，Sox 2，KLF 4和c-myc。此外，这些癌症干细胞样细胞表现出几种表达的显著增加，微RNA，最值得注意的是miR-214、miR-199、miR-10 b、miR-34 b等。转录组学和代谢组学分析表明，糖酵解速率较高，线粒体代谢，由于线粒体DNA（mtDNA）消耗这些As 3+诱导 CSC。最后，一个独特的糖酵解功能，不同于幼稚的胚胎干细胞（ESC），在这些As 3+诱导的CSC中发现癌细胞。胚胎干细胞和癌细胞都直接进行糖酵解，乳酸盐生产相反，As 3+诱导的CSC显示糖酵解的转化增加，中间体进入辅助途径，用于产生对O- 干基因的GlcNAc酰化和S-腺苷甲硫氨酸（SAM），有助于DNA 和组蛋白甲基化。因此，本申请的目标是确定：（1）是As 3+诱导的 miRNAs，特别是miR 214/199，负责线粒体DNA的消耗和随后的抑制，（2）如果是这样，那么As 3+诱导的miRNAs如何损害线粒体DNA的完整性和功能，线粒体;和（3）线粒体功能受损如何有助于产生线粒体， As 3+诱导的CSCs。我们假设As 3+诱导的JNK依赖性pSTAT 3S 727和miR-214/199 将线粒体OXPHOS转换为糖酵解以形成CSC。为了验证这一假设，提出了三个具体目标：具体目标1：As 3+激活的JNK和pSTAT 3S 727增强下调线粒体转录因子A（TFAM）的miR-214和miR-199的表达 BEAS-2B和其他肺细胞用于产生CSC。我们将着重于转录调控 miR-214/199簇，重点是启动子DNA甲基化和转录因子细胞对As 3+的反应及其下游信号传导中的结合;具体目标2：了解如何 As 3+诱导的JNK、miR-214/199和线粒体功能障碍有助于CSC的形成，强调从OXPHOS到糖酵解的代谢重编程。具体目标3：确定 As 3+诱导的JNK和miR-214/199依赖性代谢重编程在肿瘤发生中的因果作用与染色质结构相关的表观遗传学变化和与自我更新和/或通过高通量分析，观察As 3+诱导的CSC的分化。我们将鉴定代谢物- 非转化细胞、As 3+诱导转化细胞中依赖性表观遗传和染色质变化和CSC，这将通过过表达或基于CRISPR-Cas9的敲低进一步验证相关代谢途径中的关键基因，并监测自我更新和分化 CSC的地位。高通量方法将包括ChIP-seq以映射H3 K9 me 3， H3 K27 me 3和H3 K4 me 3，以及RNA-seq来分析基因的转录，特别是那些有助于CSC的多能性、自我更新和分化。我们预计这些研究的结果将揭示As 3+诱导的miR-214/199对细胞凋亡的重要性。并通过强调CSCs的生成，导致As 3+致癌的新概念的出现， As ~（3+）诱导CSC的能力。此外，我们认为，该项目产生的数据将帮助我们通过靶向JNK、miR-214/199和CSC开发新的治疗策略，利用我们独特的NOD/SCID小鼠原位肺癌模型，项目