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，连续治疗人支气管上皮细胞环境相关的浓度为3+（0.125 - 0.25毫米），一种环境金属金属，用于六个月，诱导人支气管上皮细胞的转化，其中一些具有癌症干细胞（CSC）的特征，例如体外肿瘤球体形成在体内更新，增长了干性基因的表达，包括OCT4，SOX2，KLF4和C-MYC。此外，这些癌症干细胞暴露了几个表达的明显增加 MicroRNA，最著名的是miR-214，miR-199，miR-10b，miR-34b等。此外，集成了转录组和代谢组学分析表明，糖酵解的速率更高，较低的水平这些AS3+诱导的线粒体DNA（mtDNA）部署引起的线粒体代谢 CSC。最后，一种独特的糖酵解特征，与幼稚的胚胎干细胞（ESC）和在这些AS3+诱导的CSC中发现了癌细胞。 ESC和癌细胞都指导糖酵解乳酸产生。相反，AS3+诱导的CSC显示出糖酵解的转化率增加中间体进入子公司的途径，以产生对O-重要的N-乙酰葡萄糖干性基因的Glcnacylation和S-腺苷蛋氨酸（SAM）有助于DNA 和Hisstone甲基化。彼此之间，此应用程序的目的是确定：（1）AS3+诱导 mirnas，尤其是。 miR214/199，负责mtDNA的耗竭和随之而来的抑制作用线粒体；（2）如果是这样，AS3+诱导的miRNA如何损害mtDNA和线粒体；（3）线粒体的功能受损如何有助于产生 CSC由AS3+诱导。我们假设AS3+诱导的JNK依赖性PSTAT3S727和miR-214/199 将线粒体的Oxphos切换为糖酵解，以形成CSC。为了检验这一假设，以下提出了三个具体目标：特定目标1：AS3+激活的JNK和PSTAT3S727执行 miR-214和miR-199的表达下调线粒体转录因子A（TFAM）中的表达 BEAS-2B和其他肺细胞，用于生成CSC。我们将专注于转录法规 miR-214/199群集的重点是启动子DNA甲基化和转录因子在细胞反应中与AS3+及其下游信号的结合；特定目标2：了解如何 AS3+诱导的JNK，miR-214/199和线粒体功能障碍有助于与CSC形成。强调从Oxphos到糖酵解的代谢重编程。特定目标3：定义 AS3+诱导的JNK-和miR-214/199依赖性代谢重编程的因果作用与自我更新和/或有关的与染色质结构和可及性有关的表观遗传学的变化通过高通量分析分化AS3+诱导的CSC。我们将确定代谢物 - 非转化细胞的依赖性表观遗传和染色质变化，AS3+诱导的转化细胞和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的状态。高通量方法将包括chip-seq以映射H3K9me3， H3K27me3和H3K4me3和RNA-seq谱图基因的转录，特别是。对于那些有助于CSC的多能性，自我更新和分化。我们预计拟议研究的结果将揭示AS3+诱导的miR-214/199的重要性 CSC的产生，并通过强调AS3+癌变的新概念出现 CSC诱导中AS3+的能力。而且，我们相信该项目产生的日期将通过针对JNK，miR-214/199和CSC来帮助我们制定新的治疗策略在另一项研究中，使用我们在点头/SCID小鼠中使用我们独特的小鼠骨癌模型项目。