确定组蛋白去甲基化酶抑制剂JIB-04靶向结肠癌干细胞样细胞

The goal of this research is to contribute to overcome of chemoresistance and relapse in colorectal cancer by identifying targets of histone demethylase inhibitor JIB-04 and validating its efficacy in colorectal cancer stem-like cells. we identified JIB-04 as a selective drug candidate that targets colorectal cancer stem-like cells via our preliminary study. we want to identify target proteins of JIB-04 by characterization of knockout cell lines established using CRISPR-KO system and investigate the relationship between target expression and patients’ prognosis by tissue microarray. The detailed research contents are as follows:(1)Effect of JIB-04 on cell viability of cancer stem-like cells, (2)Cloning histone demethylase genes for their overexpression, (3)Establishment of target-knockout cell lines based on colorectal cancer cell lines (HCT116 or HT-29) using CRISPR-KO system, (4)Collection of colorectal cancer tissue samples and establishment of tissue microarray (TMA) for investigation of relationship between target protein expression and patients’ prognosis, (5)Primary identification of target candidates from phenotypic screening of knockout cancer stem-like cell lines based on Oct4-reporter system, (6)Determination of driver target(s) that is responsible for survival and development of cancer stem-like cells by using tumor-sphere formation assay, (7)Identification of novel biomarker(s) for cancer stem-like cells through investigation of protein expression in colorectal cancer patient tissues.

本课题通过寻找组蛋白去甲基化酶抑制剂JIB-04的作用靶点和确定JIB-04对结肠癌样干细胞的药效的研究，有助于解决结肠癌的耐药性和复发。我们已经确定JIB-04是结肠癌干细胞样细胞的候选药物，通过对利用CRISPR-KO建立的细胞系的研究确定JIB-04作用的靶蛋白，使用组织芯片探讨靶蛋白的表达和患者预后之间的关系。研究内容包括：JIB-04对肿瘤干细胞细胞活性的影响；组蛋白去甲基化酶的克隆及表达；利用CRISPR-KO技术和结肠癌细胞株(HCT116或HT-29)建立敲出靶蛋白的细胞株；收集结肠癌组织样本，使用组织芯片，检测靶蛋白的表达和患者预后之间的关系；通过Oct4-report根据敲出靶蛋白肿瘤干细胞样细胞株的表型初步鉴定目标候选者；通过肿瘤球形成实验测定对肿瘤干细胞样细胞存活和生长起重要作用的靶蛋白；检测结肠癌患者的组织样本确定肿瘤干细胞样细胞新的生物标志物。

为了检测JIB-04是一种有效的结肠癌治疗的候选药物，我们利用MTT、qRT-PCR、western blot、肿瘤球形成实验、FACS、TOP/FOP flash荧光素酶测定和RNA测序等方法和技术，研究了用组蛋白去甲基化酶抑制剂JIB-04处理不同结肠癌细胞株后的作用效果以及作用靶点。研究结果显示：JIB-04靶向结肠癌干细胞（CSCs），能够减少三种不同结肠癌细胞系中CSCs的自我更新和干细胞特性。JIB-04处理后，在三种不同的结肠癌细胞中，结肠CSCs标志物CD133、LGR5和CD44表达减少。在体外，JIB-04明显抑制CSC肿瘤球形成，生长/复发。RNA测序分析显示JIB-04影响各种癌症相关信号通路，特别是Wnt/β-连环蛋白信号通路，这对结肠癌细胞的增殖和维持至关重要。尽管JIB-04的浓度低于沙利霉素，但JIB-04的效果等于或大于沙利霉素（已知的抗CSC药物）的效果。本研究有助于解决结肠癌的耐药性和复发，我们的研究结果强烈表明JIB-04是一种很有前景的结肠癌治疗候选药。在项目资助下发表1篇SCI、1篇核心期刊等科研论文和4篇综述（核心期刊）。培养硕士生3名，其中1名已经取得硕士学位，2名在读。同时，培养了5名临床医学专业的本科生。项目获批经费36万，支出31.808480万元，各项支出基本与预算相符。剩余经费4.191520万元，将继续用于后续研究的支出。