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

描述（由申请人提供）：膳食锌（Zn）缺乏与人类口腔食管癌的发病机制有关。在啮齿动物中，锌缺乏通过引起细胞增殖和基因表达的变化诱导舌和食管的增生表型。缺锌（ZD）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，并抑制肿瘤发生。我们的长期目标是使用我们充分表征的ZD啮齿动物癌症模型来描绘锌营养在口腔-食管癌发展和预防中的生物学作用。MicroRNA（miRNAs）是一类在转录后调节靶mRNA转录物表达的小分子非编码RNA。最近的证据表明，miRNA基因表达的改变有助于大多数人类癌症的发病机制。然而，饮食调节癌发生中的miRNA表达是一个研究不足的研究领域。我们的初步数据表明，ZD饮食诱导了不同的miRNA签名在增生ZD与锌充足（ZS）食管与显着上调的miR-31。在N-亚硝基甲基苄胺（NMBA）诱导的食管癌发生过程中，miR-31过表达在ZD食管中持续存在，具有高肿瘤结局，但在ZR食管中受到抑制，具有低肿瘤结局。重要的是，ZD大鼠体内给予锁核酸（LNA）-抗miR- 31寡核苷酸可有效地敲低食管和血液中的miR-31表达，并伴有食管细胞增殖减少。这些数据表明，miR-31表达失调可能是锌缺乏的生物学效应的机制。miR-31在几种人类癌症中过表达，包括舌鳞状细胞癌、口腔癌、肺癌、结直肠癌和肝细胞癌。有趣的是，miR-31可以利用多种机制促进肿瘤生长，但对抗乳腺癌转移。根据这些观察和我们的初步数据，我们提出了两个目标。目标1.阐明miR-31在锌缺乏驱动的食管癌前病变中的作用。我们将（a）鉴定与肿瘤发生的起始生物学相关的miR-31的下游靶基因;（B）确定miR-31的体内沉默是否通过减少细胞增殖、增加凋亡和调节经验证的miR-31靶基因的表达来减弱增生性ZD食管表型;和（c）通过比较来自LNA-抗-miR-31处理的ZD大鼠、LNA-miR-31-错配处理的ZD大鼠盐水处理的ZD大鼠和盐水处理的ZS大鼠。目标2.检验miR-31的体内敲低抵消ZD的作用并通过NMBA抑制食管肿瘤发生的假设。该提案的结果将为锌缺乏促进食管癌发生的过程提供机制见解，并促进对miR-31在癌症发展和预防中的分子作用的理解。 公共卫生相关性：鉴于膳食锌缺乏的普遍性，我们的研究确定锌如何影响食管癌发展和预防中的microRNA表达具有临床重要性。这项研究的结果将促进对锌和miR-31在癌症发展和预防中的生物学作用的理解，并确定新的microRNA种类，以改善癌症的预防，诊断和治疗。

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.