Modulation of miR-31 by dietary zinc deficiency in rat esophageal carcinogenesis

描述（由申请人提供）：膳食锌（Zn）缺乏与人类口腔癌的发病机制有关。在啮齿动物中，锌缺乏通过引起细胞增殖和基因表达的改变而诱导舌头和食道的增生性表型。缺锌大鼠对化学诱导的食管癌和舌癌非常敏感。锌补充（ZR）逆转细胞增殖，纠正异常基因表达，抑制肿瘤发生。我们的长期目标是利用我们的具有良好特征的ZD啮齿动物癌症模型，描述锌营养在口腔食管癌发展和预防中的生物学作用。MicroRNAs （miRNAs）是一类不同类型的小非编码rna，其转录后调节目标mRNA转录物的表达。最近的证据表明，miRNA基因表达的改变与大多数人类癌症的发病机制有关。然而，饮食对致癌过程中miRNA表达的调节是一个尚未充分研究的研究领域。我们的初步数据显示，ZD饮食在增生性ZD与足锌（ZS）食道中诱导了明显的miRNA信号，miR-31显著上调。在n -亚硝基甲基苄胺（NMBA）诱导的食管癌发生过程中，miR-31在肿瘤预后高的ZD食管中持续过表达，而在肿瘤预后低的ZR食管中被抑制。重要的是，在ZD大鼠体内给药锁定的核酸(LNA)-anti- miR-31寡核苷酸可有效降低miR-31在食管和血液中的表达，并伴有食管细胞增殖的减少。这些数据表明，miR-31表达失调可能是锌缺乏生物学效应的一种机制。miR-31在多种人类癌症中过表达，包括舌鳞癌、口腔癌、肺癌、结直肠癌和肝细胞癌。有趣的是，miR-31可以通过多种机制促进肿瘤生长，但反对乳腺癌转移。基于这些观察和我们的初步数据，我们提出了两个目标。目的1。阐明miR-31在缺锌驱动的食管癌前病变中的作用。我们将(a)确定miR-31的下游靶基因，这些基因与肿瘤发生的起始具有生物学相关性；(b)确定miR-31的体内沉默是否通过减少细胞增殖、增加凋亡和调节已验证的miR-31靶基因的表达来减弱增生性ZD食管表型；(c)通过比较LNA-anti-miR-31处理的ZD大鼠、lna -miR-31错配处理的ZD大鼠、生理盐水处理的ZD大鼠和生理盐水处理的ZS大鼠的食管黏膜mRNA谱，确定体内miR-31敲低后的全局基因表达变化。目标2。验证体内敲低miR-31可抵消ZD的作用，通过NMBA抑制食道肿瘤发生的假设。本研究的结果将为缺锌促进食管癌发生的机制提供深入的见解，并促进对miR-31在癌症发生和预防中的分子作用的理解。

DESCRIPTION (provided by applicant): Dietary zinc (Zn)-deficiency is implicated in the pathogenesis of human oral-esophageal cancer. In rodents Zn-deficiency induces a hyperplastic phenotype in tongue and esophagus by causing cell proliferation and changes in gene expression. Zn-deficient (ZD) rats are very sensitive to chemically-induced esophageal and tongue carcinogenesis. Zn-replenishment (ZR) reverses cell proliferation, corrects abnormal gene expression, and inhibits tumorigenesis. Our long-term goal is to delineate the biological role of Zn nutrition in oral- esophageal cancer development and prevention, using our well-characterized ZD rodent cancer models. MicroRNAs (miRNAs) are a diverse class of small non-coding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts. Recent evidence shows that alterations in miRNA gene expression contribute to the pathogenesis of most human cancers. Dietary modulation of miRNA expression in carcinogenesis, however, is an under-investigated research area. Our preliminary data show that a ZD diet induces a distinct miRNA signature in hyperplastic ZD versus Zn-sufficient (ZS) esophagus with prominent upregulation of miR-31. During N-nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis, miR-31 overexpression is sustained in ZD esophagi with a high tumor outcome but is repressed in ZR esophagi with a low tumor outcome. Importantly, in vivo administration of locked nucleic acid (LNA)-anti-miR- 31 oligonucleotide to ZD rats effectively knockdowns miR-31 expression in the esophagus and blood, accompanied by reduction in esophageal cell proliferation. These data suggest that dysregulation of miR-31 expression may be a mechanism underlying the biological effects of Zn-deficiency. miR-31 is overexpressed in several human cancers, including tongue squamous cell carcinoma, oral cancer, lung cancer, colorectal cancer, and hepatocellular carcinoma. Interestingly, miR-31 can use multiple mechanisms to promote tumor growth, but oppose breast cancer metastasis. Based on these observations and our preliminary data, we propose two Aims. Aim 1. Elucidate the role of miR-31 in Zn-deficiency driven esophageal preneoplasia. We will (a) identify downstream target genes of miR-31 that are biologically relevant to initiation of tumorigenesis; (b) determine if in vivo silencing of miR-31 attenuates the hyperplastic ZD esophageal phenotype by reducing cell proliferation, increasing apoptosis, and modulating expression of validated miR-31 target genes; and (c) identify global gene expression changes following in vivo knockdown of miR-31 by comparing mRNA profiles of esophageal mucosa from LNA-anti-miR-31 treated ZD rats, LNA-miR-31-mismatch treated ZD rats, saline- treated ZD rats, and saline-treated ZS rats. Aim 2. Test the hypothesis that in vivo knockdown of miR-31 counteracts the effect of ZD and inhibits esophageal tumorigenesis by NMBA. Results from this proposal will provide mechanistic insights into the process by which Zn-deficiency promotes esophageal carcinogenesis, as well as advance the understanding of the molecular role of miR-31 in cancer development and prevention. PUBLIC HEALTH RELEVANCE: Given the prevalence of dietary zinc-deficiency, our studies that determine how zinc influences microRNA expression in esophageal cancer development and prevention are of clinical importance. Results from the proposed research will advance the understanding of the biological role of zinc and miR-31 in cancer development and prevention, as well as identify new microRNA species, for improved cancer prevention, diagnosis, and treatment.