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）：在人胰腺癌细胞中化学预防剂异硫氰酸苄酯（BITC）介导的细胞凋亡过程中，通常观察到抗细胞凋亡蛋白Bcl-2的消耗或下调。然而，BITC 减弱 Bcl-2 表达的分子机制尚不清楚。先前的文献研究结果表明，microRNA 家族如 microRNA-16 (miR-16) 可以通过靶向 BCL2 来调节胃癌或慢性淋巴细胞白血病细胞的凋亡。 MicroRNA (miRNA) 由 19-25 个核苷酸组成，是高度保守的小非编码 RNA，可以通过在转录后水平靶向蛋白质编码基因的 mRNA 来调节发育、细胞增殖和凋亡的正常基因表达。成熟的 miRNA 能够结合 RNA 诱导的沉默复合物 (RISC)，与目标 mRNA 的 3' 非翻译区 (3' UTR) 对齐。这种相互作用导致 miRNA 的翻译抑制或裂解以及随后相应基因的下调。在此基础上，我们实验室开展了概念性新颖的研究，例如探索miRNA-16（如果有的话）在人胰腺癌细胞获得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 miR-16 上调，这可能与抗凋亡基因 BCL2 的下调有关。由于 miRNA 预计具有多个细胞靶点，我们还评估了 BITC 是否可以下调 miR-16 的另一个潜在靶点（一种促血管生成分子），如生物信息学所揭示的 FGF-2。首先，我们计划确定这些分子是否在 BITC 介导的胰腺癌细胞抗肿瘤作用中发挥重要作用。在具体目标 1 的范围内，拟议的研究将评估强制表达 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。该提案的另一个目标是评估胰腺癌细胞中miR-16的过度表达是否可以改变贴壁独立生长/细胞凋亡/血管生成，并与BITC一起使用时是否可以发挥协同作用。在这个 R21 试点提案中，我们还想探索以下目标：异硫氰酸苄酯在体内干扰 PanIN 病变发展的功效。我们的研究将旨在通过锁核酸原位杂交（LNA-ISH）分析在肿瘤微环境背景下胰腺癌早期病变的进展和消退过程中监测miRNA表达变化的程度，这有助于病理性血管生成过程。该探索性项目提出以下具体目标： 目标 1：研究异硫氰酸苄酯调节胰腺癌细胞中 miR-16 表达及其假定靶点下调的作用。目的2：探讨异位表达的miR-16 miRNA是否可以与BITC协同在胰腺癌异位异种移植模型中发挥抗肿瘤作用。目标 3：在条件性 KrasG12D 小鼠模型中评估口服 BITC 对胰腺上皮内瘤变进展过程中 miR-16 水平的影响。由于 MicroRNA 越来越多地被认为在正常和功能失调的细胞过程中发挥着至关重要的调节作用，这一新的观察表明 microRNA 与 BITC 的抗肿瘤作用有关，这应该为胰腺癌的控制提供新的见解，胰腺癌是最致命的癌症之一恶性肿瘤。 公共健康相关性：该临床前研究项目的长期目标是评估小分子量 RNA (micro RNA) 在使胰腺癌细胞对异硫氰酸苄酯（许多食用十字花科蔬菜的成分）敏感方面的作用。 MicroRNA 在正常和功能失调的细胞过程中发挥着至关重要的调节作用。它们代表了一类小的非编码 RNA 分子，已被证明与目前正在研究的几乎所有人类病理学有关。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决定，miRNA 作为细胞周期过程中的“主调节器”，其重要性正在迅速增长。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microRNA的外源表达或沉默应该对胰腺肿瘤的生长具有深远的抑制作用。这种治疗胰腺癌（最致命的恶性肿瘤之一）的策略肯定比针对单个基因更有益。