MECHANISM OF HEXAVALENT CHROMIUM CARCINOGENESIS ROLE OF LONG NON-CODING RNA DYSREGULATION

项目概要/摘要 六价铬[Cr（VI）]是一种常见的和公认的环境致癌物，可引起肺和其他疾病。 然而，在癌症中，Cr（VI）致癌的机制仍然难以捉摸。既往Cr（VI）致癌作用 研究主要集中在其遗传毒性效应。然而，研究表明，Cr（VI）暴露也会导致非 遗传毒性效应，如DNA甲基化和组蛋白调节异常所证明的表观遗传变化 翻译后修饰虽然这些观察结果为研究Cr（VI）致癌作用开辟了新的方向，但 Cr（VI）引起的表观遗传失调如何导致Cr（VI）致癌仍有待确定。 此外，对于非编码RNA（ncRNA）特别是长链非编码RNA的作用知之甚少 （lncRNA），另一种调节基因表达的表观遗传机制，在Cr（VI）致癌作用中。最近的研究 表明lncRNA是基因表达的新兴关键调节因子，并在癌症中发挥关键作用。的目标 本研究旨在探讨Cr（VI）致癌的机制，重点关注lncRNA失调的作用。 我们的初步研究发现：（i）慢性Cr（VI）暴露增加lncRNA ABHD 11-AS 1的表达， 其导致Cr（VI）诱导的细胞转化、癌症干细胞（CSC）样性质和 （ii）慢性Cr（VI）暴露下调miR-182- 5 p的表达;但敲低miR-182- 5 p的表达。 ABHD 11-AS 1增加miR-182- 5 p的水平。此外，在Cr（VI）转化细胞中过表达miR-182- 5 p， 显著降低其转化的表型，表型模仿ABHD 11-AS 1敲低的作用;（iii） 在Cr（VI）转化的细胞中Rho GT3 Rac 1和Erk 1/2 MAPK被高度激活;（iv）Rho GT3 Rac 1和Erk 1/2 MAPK的表达水平 致癌基因Rac-GEFTiam 1在Cr（VI）转化细胞中显著上调;但过表达miR- 182- 5 p或ABHD 11-AS 1的敲低大大降低了Tiam 1水平;（v）PARP-1的表达水平，这是一个关键的 DNA修复基因是基因表达的关键调控因子，在Cr（VI）转化细胞中显著上调; (vi)在Cr（VI）转化的细胞中，蛋白质PARylation水平急剧增加;抑制PARP-1或 PARP-1的敲低大大降低了蛋白质PAR化和ABHD 11-AS 1的水平， Cr（VI）转化细胞的表型。基于文献回顾和我们的新的初步数据，我们的中心 一种假说是：“慢性Cr（VI）清除上调的lncRNA ABHD 11-AS 1海绵状miR-182- 5 p，并导致其 下调，导致致癌Rac-GEF Tiam 1促进Cr（VI）水平升高 致癌作用”。提出了三个目标：目标1将确定慢性Cr（VI）暴露的机制 上调ABHD 11-AS 1。目的2将证明ABHD 11-AS 1海绵miR-182- 5 p，导致其下调， 调节和促进Cr（VI）-螯合诱导的CSC样性质、细胞转化和肿瘤发生。和 目的3将证明miR-182- 5 p的下调增加致癌Rac-GEF Tiam 1的水平， 促进Cr（VI）-螯合诱导的CSC样性质、细胞转化和肿瘤发生。Tiam 1基因敲除 将使用小鼠来证明Tiam 1在Cr（VI）诱导的肺癌发生中起重要作用。

PROJECT SUMMARY/ABSTRACT Hexavalent chromium [Cr(VI)] is a common and well-recognized environmental carcinogen causing lung and other cancers, however, the mechanism of Cr(VI) carcinogenesis remains elusive. Previous Cr(VI) carcinogenesis studies mostly focus on its genotoxic effects. However, studies showed that Cr(VI) exposure also causes non- genotoxic effects such as epigenetic changes as evidenced by dysregulations in DNA methylation and histone posttranslational modifications. While these observations open new directions for studying Cr(VI) carcinogenesis, it remains to be determined how Cr(VI)-caused epigenetic dysregulations contribute to Cr(VI) carcinogenesis. Moreover, very little is known about the role of non-coding RNAs (ncRNAs) especially the long non-coding RNAs (lncRNAs), another epigenetic mechanism regulating gene expression, in Cr(VI) carcinogenesis. Recent studies show that lncRNAs are emerging key regulators of gene expression and play critical roles in cancer. The goal of this study is to investigate the mechanism of Cr(VI) carcinogenesis focusing on the role of lncRNA dysregulation. Our preliminary studies found: (i) Chronic Cr(VI) exposure increases the expression of the lncRNA ABHD11-AS1, which contributes causally to Cr(VI)-induced cell transformation, cancer stem cell (CSC)-like property and tumorigenesis; (ii) Chronic Cr(VI) exposure down-regulates the expression of miR-182-5p; but knockdown of ABHD11-AS1 increases the level of miR-182-5p. Moreover, overexpressing miR-182-5p in Cr(VI)-transformed cells significantly reduces their transformed phenotypes, phenocopying the effect of ABHD11-AS1 knockdown; (iii) The Rho GTPase Rac1 and Erk1/2 MAPK are highly activated in Cr(VI)-transformed cells; (iv) The expression level of the oncogenic Rac-GEF Tiam1 is significantly up-regulated in Cr(VI)-transformed cells; but overexpressing miR- 182-5p or knockdown of ABHD11-AS1 greatly decrease Tiam1 level; (v) The expression level of PARP-1, a critical DNA repair gene and a key regulator of gene expression, is significantly up-regulated in Cr(VI)-transformed cells; (vi) The level of protein PARylation is drastically increased in Cr(VI)-transformed cells; inhibition of PARP-1 or knockdown of PARP-1 greatly decrease the levels of protein PARylation and ABHD11-AS1 and the transformed phenotype of Cr(VI)-transformed cells. Based on literature review and our novel preliminary data, our central hypothesis is: “Chronic Cr(VI) exposure-upregulated lncRNA ABHD11-AS1 sponges miR-182-5p and causes its down-regulation, which leads to increased level of the oncogenic Rac-GEF Tiam1 promoting Cr(VI) carcinogenesis”. Three aims are proposed: Aim 1 will determine the mechanism by which chronic Cr(VI) exposure up-regulates ABHD11-AS1. Aim 2 will demonstrate that ABHD11-AS1 sponges miR-182-5p causing its down- regulation and promoting Cr(VI)-exposure-induced CSC-like property, cell transformation and tumorigenesis. And Aim 3 will demonstrate that down-regulation of miR-182-5p increases levels of the oncogenic Rac-GEF Tiam1 promoting Cr(VI)-exposure-induced CSC-like property, cell transformation and tumorigenesis. Tiam1 knockout mice will be used to demonstrate that Tiam1 plays an important role in Cr(VI)-induced lung carcinogenesis.