Chemoprotective effects of natural products on colonic adult stem cells

描述（由申请人提供）：我们已经证明含有姜黄（姜黄素）和柑橘（柠檬苦素）生物活性的化学保护饮食可以降低慢性炎症和结肠癌的风险。在癌症生物学方面，最近的观察表明，隐窝干细胞是肠癌的起源细胞。由于非编码microrna介导的翻译抑制和染色质结构的改变可能与结肠癌的发展有关，因此确定化学预防饮食对microrna、其信使RNA （mRNA）靶点以及肠道干细胞组蛋白和DNA水平的表观遗传修饰的影响至关重要。然而，迄今为止，膳食植物药物对肠道干细胞、隐窝和肿瘤中全基因组染色质表观遗传修饰、microrna和mRNA群体的影响尚未确定。因此，我们假设含有姜黄素和/或柑橘类生物活性柠檬素的化学保护饮食将调节干细胞转录组，导致疾病进展的有利转变。Aim 1将使用高度新颖的干细胞特异性Lgr5-LacZ和Lgr-EGFP小鼠，在暴露于含有姜黄素、柠檬素及其混合物的饮食后，在结肠癌发生的起始和肿瘤阶段，量化结肠隐窝中干细胞的数量和时空位置、DNA损伤和靶向凋亡。Aim 2将使用Lgr5-EGFP小鼠研究在致癌物/炎症或生理盐水（对照）暴露后，疾病进展对结肠干细胞、完整结肠隐窝和肿瘤中microrna及其转录后调控mRNA靶点的影响。Aim 3将使用染色质免疫沉淀法为(i)肠上皮细胞隐窝和（ii）结肠肿瘤生成高分辨率全基因组“染色质状态”图，以评估饮食和结肠炎相关结肠癌发生对组蛋白和DNA水平表观遗传修饰的影响。预计新的干细胞信号网络和标记将从这些研究中出现。拟议的研究将促进我们对植物药物如何影响正常肠道发育和恶性转化过程中的关键信号通路的理解，包括干细胞的自我更新。

DESCRIPTION (provided by applicant): We have demonstrated that chemoprotective diets containing turmeric (curcumin) and citrus (limonin) bioactives reduce chronic inflammation and colon cancer risk. With respect to cancer biology, recent observations demonstrate that crypt stem cells are the cells-of-origin of intestinal cancer. Since non-coding microRNA-mediated translational repression and changes to chromatin structure may be linked to the development of colon cancer, it is vital that the effect of chemopreventive diets on microRNAs, their messenger RNA (mRNA) targets, and epigenetic modifications on both the histone and DNA levels in intestinal stem cells be determined. However, to date, the effect of dietary botanicals on genome-wide chromatin epigenetic modifications, microRNAs, and mRNA populations in intestinal stem cells, crypts and tumors has not been determined. Therefore, we hypothesize that a chemoprotective diet, containing curcumin and/or the citrus bioactive limonin, will modulate the stem cell transcriptome, resulting in a favorable shift in disease progression. Aim 1 will use highly novel stem cell specific Lgr5-LacZ and Lgr-EGFP mice to quantify the number and spatio-temporal location of stem cells, DNA damage and targeted apoptosis in the colonic crypt at the initiation and tumor stages of colon carcinogenesis following exposure to diets containing curcumin, limonin, and their mixture. Aim 2 will use the Lgr5-EGFP mouse to investigate the effect of disease progression on microRNAs and their post-transcriptionally regulated mRNA targets in colonic stem cells, intact colonic crypts and tumors following carcinogen/inflammation or saline (control) exposure. Aim 3 will generate high-resolution genome- wide "chromatin-state" maps for (i) intestinal epithelial cell crypts, and (ii) colonic tumors using chromatin immunoprecipitation in order to assess the effect of diet and colitis-associated colon carcinogenesis on epigenetic modifications at both the histone and DNA levels. It is anticipated that novel stem cell signaling networks and markers will emerge from these studies. The proposed studies will promote our understanding of how botanicals impact critical signaling pathways during normal intestinal development and malignant transformation, including self-renewal of stem cells.