Wnt通路肿瘤抑制基因去甲基化在黑树莓活性成分防治结直肠癌中的作用研究

Colorectal cancer (CRC) is one of the most common cancers in the United States. In China, it has become the third leading cause of death from cancer during the past ten years, which is consistent with observations from studies in Western populations. Epidemiological survey suggested hypomethylation on some tumor suppressor genes occurred frequently in colorectal cancer patients. Black raspberries (BRB) are a natural food enriched in protective anti-oxidant and anti-inflammatory chemicals such as ellagic acid, ferulic acid, quercetin, and anthocyanins. Previous studies have demonstrated that dietary freeze-dried BRB reduce intestinal tumor incidence and tumor development in transgenic mouse models. Data from a pilot clinical trial in colorectal cancer patients demonstrated that the chemopreventive role of BRB on colorectal tumors was associated with demethylation of relevant tumor suppressor genes. Our proposed study is designed to elucidate the roles of BRB and their bioactive components on epigenetic alterations in tumor suppressor genes associated with the Wnt-signaling pathway, both in an animal model of colitis-induced colorectal cancer and in cancer stem cells in vivo and in vitro.The specific aims are :..Specific Aim 1: Preparation of freeze-dried BRB powder and isolation and purification of BRB bioactives (anthocyanins and ellagic acid). ..Specific Aim 2: Evaluate the chemopreventive effects of freeze-dried BRB and their bioactive components in animal and cell culture model systems...Specific Aim 3: Influence of BRB active components on differentiation of colorectal cancer stem cells.

肿瘤抑制基因的过度甲基化在结直肠癌病人具有较高的发生率。黑树莓（Black Raspberry, BRB）是富含鞣花酸，花青素等酚酸类成分的天然水果。前期研究发现，BRB冻干粉对于结直肠肿瘤具有良好的预防作用，机制研究显示其预防作用与相关肿瘤抑制基因的去甲基化作用相关。本研究主要应用现代分离方法制备纯化获得BRB冻干粉、BRB花青素及BRB鞣花酸，将其分别处理AOM+DSS诱导的结直肠肿瘤大鼠动物模型及人类结直肠癌细胞，考察Wnt相关肿瘤抑制基因的表观遗传异常逆转在其肿瘤预防作用中的重要作用。进一步结合流式细胞技术对于结直肠癌干细胞群进行分选，深入探讨BRB活性成分处理前后，肿瘤干细胞中Wnt/β-catenin，Lgr5等功能基因表达及甲基化状态变化。为黑树莓的肿瘤防治作用及作用机制，和以对表观遗传异常逆转作为前景的化学预防防治靶点提供坚实的理论与实验依据。

结直肠癌 （CRC）是最常见的恶性肿瘤之一，在我国，结直肠癌发病率呈逐年增高的趋势，而治愈率并未呈现明显改善。饮食干预是能够被广为接受的预防手段用来降低结直肠癌发病率，及由此导致的死亡率。在当前研究中，化学研究部分对黑树莓冻干粉的化学成份进行了系统的分离鉴定；生物学研究发现：黑树莓花青素通过靶向炎性肠癌模型小鼠中肠道菌群紊乱的调控、表观遗传的异常逆转及对miRNA表达的调节，有效的预防及延滞AOM/DSS诱导的炎性溃疡性结肠癌的发生和发展。..1、.黑树莓冻干粉中我们通过HPLC-MS/MS分离鉴定出20个酚酸类化合物，包括5个原花青素，15个多酚。其中的10个化合物（3个花青素及7个多酚）进一步经过NMR 鉴定，这其中有2个化合物从黑树莓中首次分离得到。.2、.黑树莓花青素对于各组肠道中细菌丰富度、多样性、均匀度指数差异无显著性，说明黑树莓多酚对各组小鼠肠道菌群的种类改变无显著性作用。但是经过比对各组同种肠道菌群的数量发现，黑树莓花青素处理组与AOM/DSS模型组比较均表现出肠道益生菌（丁酸盐产生菌）升高、致病菌(弯曲杆菌、幽门螺杆菌)降低，说明黑树莓多酚可以通过改变肠道菌群的数量进而影响结直肠肿瘤的发生和发展。.3、.黑树莓花青素通过下调DNA甲基化转移酶逆转Wnt抑制基因SFRP2、WIF1启动子区域的高甲基化状态，进而降低β-catenin蛋白表达最终达到降低肿瘤细胞增殖的作用。此外，通过其他信号通路蛋白降低结肠癌的发生和转移。.4、.miRNA组织芯片结果显示：黑树莓花青素能够调控具原癌及抑癌样功能miRNA表达，深入的机制研究揭示：通过靶向调控mi-24-1-5p及其下游调控基因β-catenin及其调控的信号通路结直肠肿瘤的预防发挥重要作用。..结论：当前研究结果为黑树莓的肿瘤防治作用和作用机制及以对表观遗传异常逆转和肠道菌群异常调控作为前景的新的化学预防剂防治靶点提供可靠的理论与实验依据。