miR-34a靶向调控KLF4在结直肠癌多药耐药中的作用及机制研究

Chemotherapy multidrug resistance is the biggest problem in the therapy of advanced colorectal cancer (CRC), which involves complicated signaling pathways, and indistinct mechanism. At present, the research about miRNA and chemotherapy resistance of CRC arouse more attention. Previous studies have shown that miR-34a could inhibit proliferation, invasion, metastasis of CRC cell, and increase its sensitivity to 5-Fu by targeting regulation of KLF4. Besides, there are studies showing that miR-34a can reverse the resistance of chemotherapy in hepatocarcinoma by regulating KLF4. Therefore we propose a hypothesis that miR-34a may block the signal pathways of the drug resistance and reverse multidrug resistance in CRC by targeting KLF4. In this study, we will detect the expression of miR-34a/KLF4 in CRC tissues and try to discover their roles in chemoresistance. On this basis, we will estabilish multi-resistant CRC cell lines and xenotransplantation mice models interfered by miR- 34a/ KLF4, and monitor their tolerance to 5-Fu, L-OHP and CPT-11.What’s more, we will observe the corresponding changes of related signal pathways from different cell phenotype, molecular markers and regulatory relationships. Therefore, we could illustrate the role of miR-34a in the multidrug resistance of CRC and provide more evidence for reversing multidrug resistance and preventing the recurrence of CRC by targeting KLF4.

化疗多药耐药是中晚期结直肠癌(CRC)治疗失败的主要原因，miRNA在CRC化疗耐药的调控网络中发挥重要作用。我们前期研究发现miR-34a靶向调控KLF4可抑制CRC细胞增殖、侵袭并增强CRC 5-Fu化疗敏感性，另有研究显示miR-34a靶向调控KLF4可逆转肝癌细胞化疗耐药。由此提出假说：miR-34a通过调控靶基因KLF4的表达变化干扰CRC耐药细胞的相关信号通路,从而逆转CRC的多药耐药反应。本研究拟在检测大样本人CRC组织中miR-34a/KLF4表达，明确其与CRC化疗耐药关系的基础上，构建miR-34a/KLF4干预的CRC多药耐药细胞和动物模型，从细胞表型、分子标志物及调控关系入手，系统观察该模型对药物5-Fu、L-OHP、CPT-11耐受性及相关信号通路的变化，阐明miR-34a靶向调控KLF4在CRC多药耐药中的作用及机制,为逆转CRC多药耐药的临床问题提供科学依据。

化疗多药耐药是中晚期结直肠癌(CRC)治疗失败的主要原因，miRNA在CRC化疗耐药的调控网络中发挥重要作用。我们前期研究发现miR-34a靶向调控KLF4可抑制CRC细胞增殖、侵袭并增强CRC 5-Fu化疗敏感性，由此提出假说：miR-34a通过调控其相关靶基因的表达变化干扰CRC耐药细胞的相关信号通路,从而逆转CRC的多药耐药反应。团队4年来，一直致力于结直肠癌多药耐药复杂发病机制的研究，以关键抑癌因子miR-34a为着眼点，从转录后调控水平筛选到了KLF4、OAZ2、FOXA3为miR-34a的直接下游靶点，利用分子生物学分析手段、病理检测及体外、体内功能学实验证实miR-34a/ KLF4、miR-34a/OAZ2、miR-34a/FOXA3级联的表达、功能异常是诱导结直肠癌细胞发生多药耐药性的重要因素之一。研究证实(1)miR-34a在5-Fu化疗耐药的结肠癌组织中低表达，KLF4转录因子高表达，呈负相关，且KLF4的表达水平与患者化疗后预后呈显著负相关，提示KLF4与化疗耐药相关。上调miR-34a的表达可直接抑制KLF4的表达，提示miR-34a负向调控KLF4具有逆转结肠癌细胞化疗耐药的可能。(2)同时发现抑制内源性miR-34a的表达可促进结肠癌细胞奥沙利铂化疗耐药，机制研究发现miR-34a可以通过正向调控OAZ2信号通路抑制耐药蛋白表达逆转结肠癌细胞化疗耐药。(3)通过生信预测发现FOXA3在CRC中是一个有效的抑癌基因，FOXA3的低表达与结直肠癌多重耐药呈正相关。其可能机制为FOXA3通过抑制CRC细胞中MACC1的转录来破坏肿瘤细胞干性的维持并调节其对奥沙利铂的敏感性。因此，得出FOXA3下调介导MACC1信号通路CRC细胞癌变和耐药过程中的发挥重要功能。确定了FOXA3信号通过抑制CRC细胞干化命运来调控CRC化疗耐药性的新机制，提示FOXA3 / HDAC2 / MACC1 CRC轴或可成为CRC基因治疗的途径之一。本项目证实miR-34a可分别通过调控下游靶分子OAZ2、KLF4或FOXA3，促进CRC细胞对化疗药物抵抗性或肿瘤干细胞的激活作用，进而在CRC 肿瘤多药耐药复杂发病过程中发挥重要负性调控作用,为临床治疗多药耐药性结直肠癌提供重要的预后信息；同时对相关信号通路的后续研究筛选到多药耐药性结直肠癌个体化治疗的新靶点。