利用TFD策略研究影响胃癌阿霉素敏感性的关键转录因子亚群

Chemotherapy is a common method used on gastric cancer treatment. However, the resistance against chemotheraputic drugs significantly affects the virtue of treatment. Only about 30% efficacy was gotten when adriamycin, a frequently used chemotheraputic drug, was used singly. Drug resistance is one of the most important reasons leading to the failure of chemotherapy. Study on mechanisms of drug resistance has been focused recent years. Transcriptional factors have also been hotspot for the wide distribution, rapid and multiplex reguation they display. Nowadays most studies on transcriptional factors associated with drug resistance are limited to the function of several factors. No study is reported on these factors as groups. 31 transcriptional factors have been screened in our previous study, the expression of which showed significant difference in the gastric cancer cell line resistant to adriamycin, and the relationship of CULT1 associated with drug resistance has been demonstrated. Additionaly we found that some transcriprional factors shared one cis-element, which is called redundancy, and demostrated that some factors have similar function of inducing drug resistance. This phenomenon facilitates our work to investigate the functions of these factors as groups. In this project, transcriptional factor decoy strategy (TFD strategy) will be applied to block activity of specified factors, screen the key factor sub-group associated with sensitivity to adriamycin, and investigate the possible mechanisms which may provide experimental basis for reversion of drug resistance.

化疗是胃癌治疗的常用手段，但药物耐药的产生在很大程度上影响了化疗药物的疗效。阿霉素作为常用化疗药,单药对胃癌的有效率仅为30%，耐药是导致化疗失败的重要原因。对耐药机制的研究近年来成为关注的热点，转录因子因其作用广泛、调控迅速及方式多样成为药物耐药研究的焦点。目前耐药相关转录因子的研究多限于某几个因子的调控作用，具有耐药诱导作用的转录因子作为因子群进行研究尚无报道。我们前期利用转录因子活性谱芯片在胃癌耐药细胞系中筛选出31个具有差异活性的转录因子,并对其中的CUTL1进行了验证。分析中发现数个转录因子有时共用一个结合位点，即冗余现象，这说明某些因子具有相似的耐药诱导作用。这为我们将这些因子作为群来研究提供了便利。在本项目中,我们设想根据转录因子芯片提供的信息,通过转录因子诱饵策略阻断特定转录因子的活性, 比较耐药表型,筛选影响阿霉素敏感性的关键因子亚群，探索其机制，为逆转耐药提供实验基础。

胃癌是世界范围内的高发肿瘤，探索多药耐药机制以逆转耐药成为当前的热点之一。转录因子是一种作用广泛、调控迅速且方式多样的调控分子。在胃癌耐药的发生中发挥着重要作用。目前对转录因子的研究方法有限，寻找针对性干预转录因子的方法是阐明耐药机制的瓶颈问题。在前期研究中我们已经证实，由普通siRNA载体表达的上述种类双链RNA确实具有预期的转录因子封闭功能，并初步探讨了其发挥作用的可能机制。但尚未获得该类型双链RNA与转录因子蛋白直接相互作用的证据，其作用特异性及其在胃癌细胞中是否具有相似的功能尚不清楚。本课题将就此展开研究，并希望将其应用于胃癌耐药相关转录因子的验证中。本课题拟利用EMSA试验以获取更为直接的新型RNA结合转录因子的证据，明确新型双链RNA转录因子封闭功能的特异性，并在胃癌细胞系中对结果进行验证，同时以该新型双链RNA为工具对胃癌耐药相关因子进行验证。研究结果小结如下:. 1.EMSA结果证实，针对目的载体表达的序列所设计的探针能够与NFκB蛋白直接结合。直接地证实了目的载体发挥封闭功能并非类似于siRNA，而是类似于ODN。. 2.芯片结果显示，目的载体转染并细胞内表达所造成的基因表达变化中包括了NFκB所参与的细胞增殖、凋亡、微管形成等功能，所涉及的信号通路也多涉及NFκB。对变化较为明显的基因序列进行启动子分析，发现多数基因的调控区具有NFκB的转录因子结合位点。初步说明目的载体所产生的封闭作用具有一定的特异性。. 3.新型RNA转染后SGC7901和MKN45细胞增殖降低，凋亡增加。PCR结果显示两种细胞中NFκB的表达未发生改变，而其下游分子表达下调。说明新型RNA对胃癌细胞同样具有类似的转录因子封闭功能。. 4.针对于胃癌耐药相关转录因子共用元件的新型双链RNA分别转染SGC7901和SGC7901/ADR细胞系，并用阿霉素进行处理，初步筛选出逆转耐药效果最强的VB-ODN为VB-ODN4，针对的是AP-1相关分子；诱导耐药效果最强的是VB-ODN1针对的是AML相关分子。检测了各VB-ODN转染的细胞内经典耐药基因MDR1和MRP1的表达，并与既往文献比对，获得较为一致的结果。. 这些发现有助于提高对转录因子作用方式的认识，为胃癌多药耐药相关转录因子研究提供了新的研究工具。