JMJD2B调控干细胞活性参与ETBF介导大肠癌发生发展的机制研究

Gut microbiota dysbiosis play important roles in colorectal carcinogenesis. Dysregulated microbiome promote cancer cell stemness and involved in the development of colorectal cancer, but the mechanisms remain unknown. Histone demethylase JMJD2B is the key gene in stemness regulation. Recently, we found that Enterotoxigenic Bacteroides fragilis (ETBF) enhanced colorectal cancer cell stemness, accompanying increased JMJD2B expression. Downregulation of JMJD2B could inhibit the stemness of colorectal cancer cells. Therefore, our results suggest that JMJD2B plays an important role in ETBF-mediated stemness regulation in colorectal cancer. However, the signaling pathway responsible for JMJD2B upregulation and the molecular mechanism of JMJD2B in regulating ETBF-induced colorectal cancer stemness remain poorly understood. Therefore, based on previous work, we aim to: 1. Clarify the mechanisms of JMJD2B regulation of colorectal cancer stemness; 2. Explore the pathway of ETBF regulating JMJD2B expression; 3. Analyze the correlation of JMJD2B expression, ETBF abundance, stemness marker expression in colorectal cancer tissues, and their relationships with clinical pathological characteristics, prognosis and sensitivity to chemotherapy. These findings may provide new insights into prevention and treatment of colorectal cancer.

肠道菌群失调在促进大肠癌发生发展中发挥重要作用。肠菌失调可增加肿瘤干细胞自我更新能力，促进大肠癌的恶性进程，但具体机制不明。组蛋白去甲基化酶JMJD2B是调控干性的关键基因。最近我们发现在大肠癌中发挥重要作用的产肠毒素型脆弱拟杆菌ETBF可引起肿瘤干细胞活性增强，以及JMJD2B表达上调；抑制JMJD2B表达可降低大肠癌干细胞活性。据此我们推测JMJD2B参与ETBF介导的大肠癌干性调控，但ETBF上调JMJD2B表达的信号通路和JMJD2B调控干性的分子机制罕有研究。本研究拟在组织和细胞水平上：1、明确JMJD2B调控大肠癌干性的具体机制；2、初探ETBF调控JMJD2B表达的信号通路；3、分析大肠癌组织中ETBF丰度、JMJD2B表达与干性指标表达的相关性，以及与临床病理特征、化疗反应性和预后的关系。以揭示JMJD2B在ETBF介导的大肠癌发病中的意义，为大肠癌治疗提供理论依据。

产肠毒素脆弱拟杆菌(ETBF)在大肠癌(CRC)患者中的富集与预后不良有关。肿瘤干细胞（CSCs）在大肠癌的发生发展中起着重要的作用。但ETBF是否参与CSCs调控尚不得而知。为了阐明ETBF在干细胞活性中的作用，我们对皮下注射ETBF处理的CRC细胞的裸鼠进行了极限稀释试验(ELDA)，在ETBF灌胃后在偶氮氧甲烷(AOM)小鼠模型中培养肿瘤类器官，以及ETBF处理后CRC细胞株进行肿瘤细胞成球实验。结果表明ETBF在体内外均能提高CRC细胞的干细胞活性。此外，ETBF还增强了干细胞标记物Nanog的表达。鉴于组蛋白H3赖氨酸9三甲基化(H3K9me3)在调控CSCs特性中起着关键作用。JMJD2B作为一种表观遗传和转录调控因子，对胚胎干细胞(ESC)转化和H3K9me3去甲基化至关重要。在机制上，ETBF感染显著提高了大肠癌细胞系和裸鼠移植瘤模型中JMJD2B的水平。JMJD2B通过去甲基化NANOG启动子H3K9me3来表观遗传上调NANOG的表达，介导ETBF诱导的CRC细胞的干细胞活性。随后，我们发现ETBF激活的Toll样受体4(TLR4)途径通过活化T细胞核因子5(NFAT5)促进JMJD2B的表达。最后，在人CRC样本中，ETBF的数量与NFAT5、JMJD2B和NANOG的表达水平呈正相关。总之，ETBF在TLR4-NFAT5依赖的途径中上调JMJD2B水平，调控干细胞活性参与在大肠癌的发生发展。