转录辅激活子 SRC-3/AIB1 的泛素连接酶 FBW7 在胆管癌增殖和抗药性中的作用及机制

FBW7 plays important roles in the ubiquuitination of certain factors related with tumor, such as oncogene AIB1, and mTOR, NOTCH1 signaling et al., resulting the degradation of substrates through polyubiquitination. In our previous work, we found FBW7 mutated in cholangiocarcinoma (CC) samples and AIB1 was over-expressed in these samples. We also have confirmed that SRC-3/AIB1 could enhance human CC cell proliferation and chemoresistance. Our hypothesis is that the mutation of FBW7 in CC would lead to the functional deficiency of itself and high expression of AIB1, promoting the proliferation and drug resistance of CC. First, we would analyze the mutation situation of FBW7 and the expression level of FBW7, AIB1,mTOR,NOTCH1 in CC samples by immunohistochemistry， fluorescent quantitation PCR and western blot ; Then, we would undertake the technology of over-expression and gene silence to study the impact of FBW7 on CC cell in the respects of proliferation and drug resistence; Thirdly, we would explore the interaction between FBW7 and AIB1, mTor, NOTCH1 signaling. At last, we are going to confirm if FBW7 could regulate the level of AIB1, mTor or NOTCH1, leading proliferation and chemoresistance of CC, by which to explore the function and mechanism of FBW7 in the proliferation and chemoresistance of CC. Our study would enrich the signal pathway information of CC cell, providing some novel ideal on safe and effective therapeutic targets of CC.

泛素连接酶FBW7常泛素化肿瘤相关因子，如原癌基因AIB1和mTOR、NOTCH1等，导致底物降解。我们前期研究发现，胆管癌样品中FBW7高突变，且伴有 AIB1 高表达，同时AIB1还可促进胆管癌细胞增殖和抗药性。我们假设 FBW7 突变致其功能缺失，不能泛素化AIB1等，使之高表达，促进细胞的增殖和抗药性。本项目拟首先检测样品中FBW7的突变情况，通过荧光定量PCR、免疫组化等检测样品中FBW7、AIB1、mTOR、NOTCH1等的表达；然后运用过表达、基因沉默研究FBW7的表达变化对胆管癌细胞增殖和抗药性的影响；最后探讨FBW7对AIB1的作用，及其与mTOR和NOTCH1通路的关系，论证FBW7通过泛素化AIB1、mTOR或NOTCH1等影响胆管癌细胞增殖和抗药性，揭示FBW7在胆管癌增殖和抗药性中的作用和分子机制，丰富胆管癌细胞信号通路信息，为寻找更为安全有效的治疗靶点提供思路。

(一）FBW7属于SCF（complex of SKP1, CUL1 and F-box protein）型E3泛素连接酶底物识别组分。由于FBW7可以以多个促癌基因为底物以及其在多种肿瘤中发生突变，因此被广泛认为是一种抑癌因子，然而FBW7在胆管癌中的作用并不清楚。我们发现过表达FBW7α可以诱导胆管癌细胞停滞在G1期，并且抑制细胞的增殖以及裸鼠移植瘤的生长。进一步研究发现过表达FBW7α促进c-Myc和cyclinE蛋白的降解，恢复c-Myc蛋白的表达增加过表达FBW7α细胞的增殖，但恢复cyclinE的表达却不影响FBW7过表达细胞的增殖。我们的结果表明FBW7α-cMyc信号通路在胆管癌发展进程中发挥着重要的作用，作为FBW7底物的c-Myc可能是治疗胆管癌的潜在重要靶点。另外，我们发现过表达FBW7α减少胆管癌对顺铂的敏感性，并且减少DNA的损伤。.（二）柯里拉京属于天然植物多芬单宁酸类化合物，具有抗炎，抗氧化和抗肿瘤的作用，然而其机制并不是太清楚，我们发现柯里拉京可以抑制胆管癌细胞的增殖，迁移和侵袭，并且促进细胞凋亡，与这些表型相伴随的是柯里拉京抑制Notch信号通路。在裸鼠中，柯里拉京同样抑制胆管癌移植瘤的生长和Notch信号通路。这些研究表明柯里拉京可能昨晚一种潜在的治疗胆管癌的药物。