转录因子AhR靶向ABCG2和VEGF-B对绒癌干细胞样特性影响的研究

Choriocarcinoma is a common tumor in women of fertile age,it can transfer to the whole body through blood at early stage . Unlike other tumors, Choriocarcinoma is more sensitive to chemotherapy,and may be cured by chemotherapy .However, in recent years the drug resistance of choriocarcinoma has being increasingly severe, which makes it as the main reason for the failure of choriocarcinoma treatment.Research shows that the existence of cancer stem cells may be an important reason for tumorigenesis,drug resistance and metastasis . In the pre-experiments,our research group has successfully isolated choriocarcinoma stem-like cells from choriocarcinoma cell lines and found its high expression of ABCG2; Before,we also found the drug-resistant choriocarcinoma cells highly express VEGF - B. Recent studies show that aryl hydrocarbon receptor AhR is closely related to the tumorigenesis, cancer drug resistance and metastasis; Studies confirm AhR can increase the expression of ABCG2, and we have found that the startup sequence of VEGF - B exist AhR binding site,but have not yet found research reports about AhR 's role in the regulation of the expression of VEGF - B Our team will centre on AhR 's role in the regulation of the expression of VEGF - B and ABCG2,as well as its effect on cancer stem cell-like properties of choriocarcinoma, we'll be first to explore the interaction of AhR and VEGF - B, research about AhR's influernce on choriocarcinoma growth, dtug resistance ,metastasis and the mechanism, expectingt to put forward a new clue to choriocarcinoma's treatment.

绒毛膜癌（绒癌）是发生于育龄女性的常见高度恶性肿瘤，早期即可血行转移至全身。与其他肿瘤不同，绒癌对化疗比较敏感，并可通过化疗治愈。但近来绒癌耐药日益加重，成为其治疗失败的主要原因。研究表明肿瘤干细胞的存在可能是肿瘤发生、耐药、转移的重要原因。本课题组前期已成功从绒癌细胞中分离出绒癌干细胞样细胞，发现其高表达ABCG2；前期试验也发现绒癌耐药细胞高表达VEGF-B。近来研究表明芳基烃受体AhR与肿瘤发生、耐药、转移密切相关；研究证实AhR可上调ABCG2的表达，且我们发现VEGF-B启动序列上也存在着AhR的结合位点，但尚未见关于AhR对VEGF-B表达调控的研究报导。本课题将以AhR对VEGF-B、ABCG2的表达调控及对绒癌干细胞样特性的影响为主线，率先探讨AhR与VEGF-B的相互作用，并研究AhR靶向ABCG2和VEGF-B对绒癌生长、耐药、转移的影响和机制，有望提出绒癌治疗的新线索

目的：绒癌是发生于生育年龄妇女的常见高度恶性肿瘤，绒癌细胞转移到脑是绒癌致死的主要原因之一。绒癌治疗以化疗为主，近年来耐药绒癌病例不断增多，对化疗耐药是绒癌治疗失败的主要原因。肿瘤干细胞（CSCs）是存在于肿瘤组织中一类数目极少但具有干细胞特性的细胞亚群，具有自我更新能力和不定向分化潜能，有多药耐药和放化疗抵抗的特点。我们以绒癌JEG-3细胞和绒癌干细胞样细胞为模型，以转录因子AhR为关键节点，验证VEGF-B和ABCG2是否是AhR的靶基因并进一步对AhR与VEGF-B和ABCG2的靶向关系进行研究。为寻找减弱或逆转绒癌耐药、阻断其侵袭转移的有效方法提供新的靶点和依据。.方法：无血清悬浮培养肿瘤球形成试验、MTS/CCK8法、克隆形成实验、划痕实验、Transwell实验、流式细胞术、裸鼠皮下成瘤实验鉴定干细胞样特性及探讨AhR在绒癌侵袭转移、耐药中的作用及对其干细胞样特性的影响，报告基因和免疫共沉淀验证AhR对VEGF-B、ABCG2的调节作用。 .结果：1. 悬浮培养筛选出悬浮生长、可连续传代的微球体，高表达Oct-4、Sox-2、Nanog、CD133、CD44，较亲本细胞具有更高的克隆形成率、细胞增殖率、迁移率和透膜细胞数和化疗药物MTX、VP16作用下的细胞存活率。. 2.绒毛膜癌干细胞样细胞相对高表达转录因子AhR， 改变AhR表达及活性影响绒癌细胞细胞增殖率、肿瘤干细胞形成率、迁移率和透膜细胞数、移植瘤体积及化疗药物MTX、VP16作用下的细胞存活率。. 3.绒癌干细胞样细胞中VEGFB、ABCG2表达相对升高， ABCG2可以影响绒癌JEG-3细胞的肿瘤干细胞样细胞形成率；VEGFB可以影响绒癌JEG-3细胞的肿瘤干细胞样细胞迁移和侵袭细胞数。. 4.AhR可与VEGFB、ABCG2基因启动子区结合。.结论：1.无血清悬浮培养法可获得绒癌干细胞样细胞；2. 绒癌肿瘤干细胞样细胞具有更高的克隆形成能力、细胞增殖能力、对化疗药物MTX、VP16的抵抗能力、侵袭转移能力及体外成瘤能力。3.绒癌干细胞样细胞较亲本细胞高表达AhR，改变AhR表达及活性影响绒癌细胞干细胞样细胞特性。4. AhR是通过直接与VEGFB、ABCG2基因启动子区结合进而参与对其表达调控。

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