KLF4对宫颈癌细胞放疗敏感性的调节作用及分子机制

Because of the existence of radidiotherapy resistance,the treatment effect of the cervical cancer is still not satisfied,while the adjustment mechanism of radidiosensitivity is still not well known. We previous study resultes indicated that tumor-suppression gene NDRG2 could adjust cervical cancer cells' radidiosensitivity through invoving in the stress response after DNA damage,and the transcription factor KLF4 could activate NDRG2 by transcription function. Based on literatures, we imply that KLF4 may play a role in the adjustment mechanism of cervical cancer cells' radidiosensitivity. Based on the previous studies, we'll make further clear that wether KLF4 is concerned with radidiosensitivity of the cervical cancer cells. Using DNA recombination technology we'll construct KLF4 gene expression plasmid vector and KLF4 gene siRNA plasmid vector and by transfecting the both plasmid vetor into cervical cancer cells, we'll acquired the stable transfected cell lines, which has different KLF4 expression status.Thus,we'll make clear that in what manner KLF4 could adjust cervical cancer cells' radidiosensitivity through in vitro and in vivo studies. Furthermore,by means of gene chip and chromatin immunoprecipitation assay, we'll detect differential gene expression after modulation of KLF4 expression; by means of bioinformatics technology we'll verify and analysis the interaction factors of KLF4 and the possible mechanism. In the whole,it plays importannt theoretical and practical role in seeking new molecular targets and further exploring new radiation sensitization strategies for cervical cancer treatment by our work.

由于放疗抵抗的存在，宫颈癌的治疗效果仍然受限，放疗敏感性的调节机制仍未完全阐明。我们的前期研究工作证实：转录因子KLF4在宫颈癌的发生发展过程中发挥抑癌基因作用，对抑癌基因NDRG2具有转录激活作用，不仅与宫颈癌细胞放疗敏感性相关而且还参与了细胞受照射后DNA损伤诱发的应激反应。结合文献我们推测：KLF4可能作为NDRG2上游调控元件参与DNA损伤诱发的应激反应调节宫颈癌细胞放疗敏感性。本课题拟在前期研究基础上，利用DNA重组技术构建KLF4的正义表达载体及针对KLF4的siRNA表达载体，借助细胞转染、RNAi技术改变KLF4表达状况，通过体内外实验明确KLF4对宫颈癌细胞放疗敏感性的调节作用；采用基因芯片、染色质免疫共沉淀检测KLF4表达调变后差异基因表达，生物信息学技术验证分析KLF4相互作用分子，对进一步探讨宫颈癌放疗增敏新途径，寻找新的分子靶标具有重要的理论与实际意义。

由于放疗抵抗的存在，宫颈癌的治疗效果仍然受限，放疗敏感性的调节机制仍未完全阐明。我们的前期研究工作证实：转录因子KLF4( Krüppel-like factor 4)在宫颈癌的发生发展过程中发挥抑癌基因作用，结合文献我们推测：KLF4可能参与DNA损伤诱发的应激反应调节宫颈癌细胞放疗敏感性。本课题从组织、细胞、动物以及分子机制4个方面探讨了宫颈癌放疗增敏新途径。①回顾性病例研究显示：KLF4表达与放疗敏感性相关，在放疗耐受组中表达高于敏感组，KLF4的高表达可能与放疗后原位复发相关而与远处转移无关；KLF4高表达组总生存期和无瘤生存期显著低于低表达组；多因素COX回归分析提示：KLF4高表达和组织类型可能是影响预后的高风险因素。②选择KLF4高表达的HeLa和低表达的C33A作为后续实验细胞株，通过平板克隆，细胞流式术检测周期和凋亡鉴别HeLa和C33A细胞放疗敏感性，使用慢病毒制备KLF4不同表达状况的宫颈癌细胞株，结果显示：在宫颈癌细胞中，KLF4与放疗敏感性相关，KLF4表达增高降低宫颈癌细胞放疗敏感性。③通过在体模型确认KLF4对宫颈癌裸鼠移植瘤生长及放疗敏感性的调节作用，证实KLF4高表达降低裸鼠移植瘤放疗敏感性。④运用表达谱基因芯片检测，筛选差异基因，KLF4上调15个基因，而与放疗相关的基因只有胰岛素样生长因子2（insulin-like growth factor2, IGF2），我们进一步研究发现KLF4可能通过上调IGF2激活AKT途径降低宫颈癌放疗敏感性。上述实验结果对进一步探讨宫颈癌放疗增敏调节机制，寻找新的分子靶标具有重要的理论与实际意义。