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

我们已经证明，用人支气管上皮细胞连续处理 与环境有关的As3(0.125-0.25M)浓度，这是一种环境类金属，用于 六个月，诱导人支气管上皮细胞转化，其中一些拥有 肿瘤干细胞(CSCs)的特性，如体外形成肿瘤球体，自体 体内更新，增加了包括Oct4、Sox2、KLF4和c-myc在内的茎基因的表达。 此外，这些癌症干细胞样细胞表现出明显增加的几个 MicroRNAs，最显著的是miR-214、miR-199、miR-10b、miR-34b等。 转录组和代谢组分析显示糖酵解率较高，而血浆中 As3诱导的这些细胞中线粒体DNA(MtDNA)耗竭所致的线粒体代谢 CSCS。最后，一个独特的糖酵解特征，不同于幼稚的胚胎干细胞(ESCs)和 在As3诱导的CSCs中发现了癌细胞。胚胎干细胞和癌细胞均可直接糖酵解 乳酸盐生产。相反，As3诱导的CSCs表现出糖酵解的转化增加 中间体进入生成N-乙酰氨基葡萄糖的辅助途径，对O- 茎秆基因的GlcN酰化和对DNA有贡献的S腺苷蛋氨酸 和组蛋白甲基化。因此，本申请的目标是确定：(1)是否由As3诱导 MiRNAs，尤指MiR214/199，负责线粒体DNA的耗尽和随之而来的抑制 (2)如果是这样，As3诱导的miRNAs如何损害mtDNA的完整性和功能，以及 线粒体；以及(3)线粒体功能受损如何导致 As3诱导的CSCs。我们假设As3诱导依赖JNK的pSTAT3S727和miR-214/199 将线粒体OXPHOS转换为糖酵解以形成CSCs。要检验这一假设，请看以下内容 提出了三个具体目标：具体目标1：As3激活的JNK和pSTAT3S727的执行 下调线粒体转录因子A(TFAM)的miR-214和miR-199的表达 BEAS-2B等肺细胞用于CSCs的生成。我们将重点关注转录调控。 以启动子DNA甲基化和转录因子为重点的miR-214/199簇 细胞对As3及其下游信号反应的结合；特定目标2：了解如何 As3诱导的JNK、miR-214/199和线粒体功能障碍参与了CSCs的形成 强调从氧磷酸盐到糖酵解的代谢重新编程。具体目标3：界定 As3诱导的JNK和miR-214/199依赖的代谢重编程在脑缺血再灌注损伤中的因果作用 与染色质结构和可及性相关的与自我更新和/或相关的表观遗传学变化 通过高通量图谱鉴定As3诱导的CSCs。我们将鉴定代谢物- As3诱导的转化细胞中未转化细胞的表观遗传学和染色质依赖性变化 和CSCs，将通过过度表达或基于CRISPR-Cas9的基因敲除进一步验证 相关代谢途径中的关键基因与自我更新和分化的监测 CSC的状态。高通量方法将包括CHIP-SEQ到MAP H3K9me3， H3K27me3和H3K4me3，以及RNA-seq来分析这些基因的转录，特别是。对于那些 促进CSCs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在 CSCs的产生，并通过强调As3致癌的新概念而导致As3致癌新概念的出现 As3诱导CSC的能力。此外，我们相信，该项目产生的日期将 通过以下途径帮助我们开发针对JNK、miR-214/199和CSCs的新治疗策略 在另一项研究中利用我们独特的NOD/SCID小鼠原位肺癌模型 项目。

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.