p53—miR-509-5p—Mdm2—p53反馈环路影响宫颈癌细胞生长的机制研究

MiRNAs are a class of regulatory short non-coding RNAs. In the present study, we focused on the identification of the miRNAs which were involved in the p53 tumor suppressor regulatory pathway in cervical cancers. Our previous resluts have showed that wild-type p53 increased the expression of miR-509-5p at the transcription level. Meanwhile, our studies demonstrated that, in cervical cancer HeLa cells carrying wild-type p53, miR-509-5p induced cell cycle arrest and apoptosis, inhibited cell growth and proliferation, functioning as a tumor suppressor gene. In addition, we confirmed that miR-509-5p inhibited the expression of Mdm2 through directly binding the 3' UTR of its mRNA, and upregulated their downstream moleculars, including p53, p21 and bak, thus contributing to a positively regulatory feedback loop of p53-miR-509-5p-Mdm2-p53. We will go on evaluating the effets of miR-509-5p on the growth and proliferation of cervical cancer cells using the rescue assays, in vivo tumorigenesis assays in combination with the analysis of clinical tissues. Furthermore, we will identificate the core promoter of miR-509-5p and its transcription start site, and investigate the detail regulatoy mechanism through which miR-509-5p was upregulated by wild-type p53 using EGFP reporter assays, EMSA, ChIP assays etc. All of above these will be helpful to understand the human cervical cancer biology, and provide novel clues for the diagnosis and treatment of cervical cancer.

miRNA是一类调节性小RNA分子。本项目立足寻找宫颈癌中参与p53肿瘤抑制途径的miRNA。前期工作证实野生型p53在转录水平诱导miR-509-5p表达增加，而且在野生型p53的宫颈癌HeLa细胞中，miR-509-5p诱导细胞周期停滞和凋亡增加，抑制细胞生长，发挥抑癌基因作用。并且证实miR-509-5p通过直接靶定并下调Mdm2分子，进而上调p53、p21及bak表达，形成了p53-miR-509-5p-Mdm2-p53正反馈调控环路。我们将进一步应用挽救实验、裸鼠动物体内实验及临床标本研究，完善miR-509-5p对宫颈癌细胞生长增殖的影响。同时，寻找miR-509-5p的核心启动子及转录起始位点，并结合荧光报告载体、EMSA及ChIP等实验深入分析野生型p53对miR-509-5p转录调控的直接性，从而为宫颈癌发生发展的分子机制提供理论基础，并为宫颈癌的诊断和治疗提供有力线索。

微小RNA（miRNA）是一大类约22nt大小的高度保守的非编码单链RNA分子，对细胞内基因表达进行转录后调控。研究已经证实，miRNA参与了包括肿瘤发生发展在内的多种生理及病理过程。肿瘤抑制蛋白p53是细胞内非常重要的的抑癌基因，通过转录调控下游基因如鼠双微基因Mdm2、p21和bax等来抑制细胞周期或促进细胞凋亡，进而发挥抑癌基因的作用。同时，Mdm2蛋白又可以与p53特异性结合，抑制其反式激活功能或促进p53蛋白的降解，从而对p53蛋白进行负反馈调控，形成p53和Mdm2之间的精细平衡状态。本项目在研究中发现并证实，p53与Mdm2之间除了直接相互的调控作用外，还可以通过miR-509-5p来进行间接调控：p53可以直接结合在miR-509-5p上游的启动子区域，在转录水平上调其表达；表达增加的miR-509-5p则结合其下游直接靶基因Mdm2的3’UTR区进而抑制其mRNA表达及翻译。Mdm2表达水平的降低直接解除了其对p53蛋白的负反馈作用，使得p53表达增加，进而导致p53下游直接靶基因p21及bak的表达增加，从而诱导细胞周期停滞及细胞凋亡。同时，我们还发现，在宫颈癌及肝癌组织中，p53和miR-509-5p表达水平明显偏低，而Mdm2则明显高于其邻近正常组织，而且进一步细胞功能学实验证实miR-509-5p可以抑制宫颈癌和肝癌细胞的生长增殖以及侵袭与迁移能力，同时诱导细胞凋亡及细胞周期停滞于G1期，发挥抑癌基因作用，并且进一步证实这一过程是通过抑制其下游直接靶基因Mdm2实现的。总之，本研究发现miR-509-5p参与了Mdm2/p53通路的调节，并参与了宫颈癌及肝癌的的发生发展过程，为肿瘤发生发展的分子机制提供了研究线索。