Regulation of microRNA-16 by benzyl isothiocyanate in pancreatic cancer

异硫氰酸苄酯对胰腺癌中 microRNA-16 的调节

• 批准号: 8096691
• 负责人: SUBRATA HALDAR
• 金额: $ 16.56万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8096691
• 关键词: 3' Untranslated Regions; Adenocarcinoma Cell; Affect; Anchorage-Independent Growth; Animal Feed; Apoptosis; Apoptotic; Asses; Attenuated; BCL2 gene; Binding; Bioinformatics; Biological Assay; Biological Process; Biology; CDKN1C gene; Cell Cycle; Cell Death; Cell Line; Cell Proliferation; Cell physiology; Cells; Chemopreventive Agent; Chronic Lymphocytic Leukemia; Clinical; Code; Data; Development; Down-Regulation; FGF2 gene; Family; Fibroblast Growth Factor 2; Functional RNA; Future; Gene Expression; Gene Targeting; Genes; Goals; Human; Human Pathology; Immune response; Immune system; Immunocompromised Host; In Situ Hybridization; In Vitro; Individual; Intervention; Investigation; Isothiocyanates; Laboratories; Lesion; Link; Literature; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Messenger RNA; MicroRNAs; Molecular; Monitor; Nucleotides; Nude Mice; Oncogenic; Oral Administration; Pancreas; Pancreatic Adenocarcinoma; Pancreatic Ductal Adenocarcinoma; Pancreatic Intraepithelial Neoplasia; Pancreatic carcinoma; Pathway interactions; Play; Primary Lesion; Process; Prostate; Proteins; Publishing; RNA-Induced Silencing Complex; Regulation; Regulator Genes; Reporting; Research; Research Project Grants; Research Proposals; Resistance; Role; Small Nuclear RNA; Stomach; Translating; Translational Repression; Tumor Burden; Tumor Suppressor Proteins; Untranslated RNA; Untranslated Regions; Up-Regulation; Viral; Xenograft Model; angiogenesis; base; benzyl isothiocyanate; cancer cell; cellular targeting; cruciferous vegetable; in vitro Model; in vivo; in vivo Model; insight; locked nucleic acid; malignant stomach neoplasm; mouse model; novel; novel therapeutics; overexpression; pancreatic cancer cells; pancreatic neoplasm; pre-clinical; pre-clinical research; programs; public health relevance; stem cell fate; tool; trait; tumor; tumor growth; tumor progression; tumor xenograft; tumorigenesis; vector

DESCRIPTION (provided by applicant): The depletion or down modulation of antiapoptotic protein Bcl-2 is commonly observed during apoptosis mediated by chemopreventive agent benzyl isothiocyanate (BITC) in human pancreatic cancer cells. However, the molecular mechanism by which BITC can attenuate Bcl-2 expression is not clear. Previous findings in the literature suggest that microRNA family such as microRNA-16 (miR-16) could modulate apoptosis in gastric cancer or chronic lymphocytic leukemia cells by targeting BCL2. MicroRNAs (miRNAs) comprising of 19-25 nucleotides, are highly conserved small non-coding RNAs which can regulate normal gene expression for development, cell proliferation and apoptosis by targeting mRNAs of protein coding genes at the posttranscriptional level. Mature miRNAs are capable of binding the RNA-induced silencing complex (RISC) to align with target mRNAs at their 3' untranslated region (3' UTR). Such interaction leads to translational repression or cleavage of miRNAs and the subsequent down-modulation of the respective genes. On this basis, conceptually novel studies, such as exploring the role of miRNA-16 (if any) in the acquisition of BITC sensitivity of human pancreatic cancer cells, were undertaken in our laboratory. Interestingly, pancreatic cancer cells treated with chemopreventive agent BITC revealed up regulation of miR-16 which might be linked to down modulation of antiapoptotic gene BCL2. Since miRNAs are expected to have multiple cellular targets, we also assessed whether BITC can down regulate another potential target of miR-16 a proangiogenic molecule, FGF- 2 as revealed by bioinformatics. Primarily, we plan to determine whether these molecules play important roles in BITC mediated antitumor effect on pancreatic cancer cells. Under the scope of Specific aim 1, the proposed studies will asses whether forced expression Bcl-2 or FGF-2 (lacking 32-UTR region) can rescue cell death and angiogenesis mediated by BITC. Another goal of this proposal is to assess whether overexpression of miR-16 in pancreatic cancer cells can alter anchorage independent growth /apoptosis/angiogenesis and can exert synergistic effect when employed together with BITC. In this R21 pilot proposal, we also like to explore the following objective: the efficacy of benzylisothiocyanate to interfere with the development of PanIN lesions in vivo. Our investigation will be directed to monitor the magnitude of miRNA expression changes by locked nucleic acid in situ hybridization (LNA-ISH) analysis during the progression and regression of early lesions of pancreatic cancer in the context of tumor microenvironment which contributes to the pathological angiogenic process. The following specific aims are proposed for this exploratory project: Aim 1: To investigate the role of benzyl isothiocyanate modulated miR-16 expression and down regulation of its putative targets in pancreatic cancer cells. Aim 2: To investigate whether ectopically expressed miR-16 miRNA can cooperate with BITC to exert anti tumor effect in heterotopic xenograft models of pancreatic cancer. Aim 3: To evaluate the effect of orally administered BITC on the level of miR-16 during the progression of Pancreatic Intraepithelial Neoplasia in a Conditional KrasG12D mouse model. Since MicroRNAs, are increasingly being accepted as playing a crucial regulatory role in normal and dysfunctional cellular processes, this novel observation implicating microRNAs with the antitumor effect of BITC should provide new insights in regard to the control of pancreatic cancer, one of the deadliest of all malignancies. PUBLIC HEALTH RELEVANCE: The long-term goal of this preclinical research project is to assess the role of small molecular weight RNA (micro RNA) in rendering pancreatic cancer cells sensitive to benzyl isothiocyanate, a constituent of many edible cruciferous vegetables. MicroRNAs play a crucial regulatory role in normal and dysfunctional cellular processes. They represent a class of small, noncoding RNA molecules, which have been shown to be involved in almost every human pathology currently under study. From tumor progression and viral host interactions, to immune response and stem cell fate determination, miRNAs are quickly growing in importance as the "master regulators" in cell cycle processes. Given that microRNAs can have multiple target genes and can concurrently decrease the levels of genes with different biological functions, the exogenous expression or silencing of microRNAs should have profound inhibitory effect on pancreatic tumor growth. This strategy for tackling pancreatic cancer, one of the deadliest malignancies of all, is definitely more beneficial than targeting individual genes.

描述（由申请人提供）：抗凋亡蛋白Bcl-2的耗竭或下降调节在人类胰腺癌细胞中由化学预防剂苄基异硫氰酸苯甲酸苯甲酸苯甲酸苯甲酸苯甲酸酯（BITC）介导的凋亡中通常观察到。但是，BITC可以减弱Bcl-2表达的分子机制尚不清楚。文献中先前的发现表明，microRNA家族（例如microRNA-16）可以通过靶向BCL2来调节胃癌或慢性淋巴细胞性白血病细胞的凋亡。由19-25个核苷酸组成的microRNA（miRNA）是高度保守的小型非编码RNA，可以通过靶向在转录后水平上靶向蛋白质编码素质的mRNA来调节正常基因表达以进行发育，细胞增殖和凋亡。成熟的miRNA能够结合RNA诱导的沉默复合物（RISC），与3'未翻译区域（3'UTR）处于目标mRNA。这种相互作用导致miRNA的翻译抑制或切割以及随后对各个基因的下调。在此基础上，我们的实验室进行了概念上的新研究，例如探索miRNA-16（如果有的话）在获得人类胰腺癌细胞的BITC敏感性中的作用。有趣的是，用化学预防剂BITC处理的胰腺癌细胞显示了miR-16的调节，这可能与抗凋亡基因BCl2的调节有关。由于预期miRNA具有多个细胞靶标，因此我们还评估了BITC是否可以降低MiR-16的另一个潜在靶标的促血管生成分子，即FGF-2，如生物信息学所示。首先，我们计划确定这些分子是否在BITC介导的抗肿瘤作用对胰腺癌细胞中起重要作用。在特定目标1的范围下，拟议的研究将评估强迫表达Bcl-2或FGF-2（缺少32-UTR区域）是否可以挽救BITC介导的细胞死亡和血管生成。该建议的另一个目标是评估胰腺癌细胞中miR-16的过表达是否可以改变锚定独立的生长 /凋亡 /血管生成，并在与BITC一起使用时会发挥协同作用。在此R21试点建议中，我们还喜欢探索以下目标：苄基异氰酸酯的功效干扰体内Panin病变的发展。我们的研究将指示通过在肿瘤微环境的背景下，在胰腺癌早期病变的进展和回归过程中，通过锁定的原位杂交（LNA-ish）分析来监测miRNA表达的变化（LNA-ish）分析，这有助于病理血管生成性过程。为此探索性项目提出了以下特定目的：目的1：研究苄基异硫氰酸酯调制的miR-16表达和胰腺癌细胞中假定靶标的作用。目标2：研究异位表达的miR-16 miRNA是否可以与BITC合作以在胰腺癌的异位异种移植模型中发挥抗肿瘤效应。目标3：评估在条件KRASG12D小鼠模型中，在胰腺内肿瘤进展过程中，口服施用BITC对miR-16水平的影响。由于microRNA在正常和功能障碍的细胞过程中扮演着关键的调节作用，因此越来越多地被接受，因此这种新的观察结果牵涉到MicroRNA和BITC的抗肿瘤作用，应就控制胰腺癌的控制提供新的见解，这是所有恶性肿瘤中最致命的一项。 公共卫生相关性：该临床前研究项目的长期目标是评估小分子量RNA（微RNA）在使胰腺癌细胞敏感的胰岛异硫氰酸酯敏感的胰腺癌细胞中的作用，这是许多可食用的十字杂交蔬菜的组成部分。 microRNA在正常和功能失调的细胞过程中起关键的调节作用。它们代表了一类无编码的RNA分子，这些分子已证明与目前正在研究的几乎所有人类病理有关。从肿瘤进展和病毒宿主相互作用到免疫反应和干细胞命运的确定，miRNA在细胞周期过程中的“主要调节剂”的重要性迅速增长。鉴于microRNA可以具有多个靶基因，并且可以同时降低具有不同生物学功能的基因的水平，因此，微生物的外源表达或沉默应对胰腺肿瘤生长具有深远的抑制作用。解决胰腺癌的这种策略是最致命的恶性肿瘤之一，绝对比靶向单个基因更有益。