DNA解旋酶亚基MCM7调控端粒酶hTERT活性的分子机制研究

MCM7 is a subunit of MCM2-7 complex which plays an important role in the initiation of DNA replication as a DNA helicase during cell division. However, the majority of MCM7 is presented as a free form but its function is not well understood. It has been reported that MCM7 protein is localized in telomere, but its biological significance is not clear. Our preliminary data indicated that MCM7 can promote the telomerase activity while knockdownof MCM7 can cause the decrease of telomerase activity. Interestingly,we found that MCM7 can interact with telomerase catalytic subunit hTERT and also enhance the stability of hTERT protein. In order to investigate the mechanism of MCM7 in up-regulating the activity of hTERT and whether MCM7 can promote tumorigensis by influence telomerase activity, we plan to accomplish the following studies: (1) To identify the binding site or domain of MCM7 with hTERT, and examine whether MCM7 affects the association of hTERT and human telomerase RNA template (hTR); (2) It is known that phosphorylation of MCM7 by cyclin E/Cdk2 affects its chromatin localization. Therfore we will investigate whether the phosphorylation of MCM7 influences its function on regulating the subcellular localization of hTERT as well as its stability; (3) To establish the MCM7 and its mutant inducible cell lines and examine whether MCM7 promotes the cell proliferation,migration and tumorigenesis by up-regulating hTERT using mice model. (4) To study the clinical correlation of MCM7 and hTERT in hepatocarcinoma samples and reveal the relevance of MCM7 and hTERT in tumorigenesis as well as prognosis of HCC.

MCM7是DNA解旋酶MCMs亚基之一，在DNA复制起始过程中发挥重要作用，有研究提示MCM7具有不依赖于MCMs的功能，我们前期研究发现MCM7具有提高端粒酶活性的作用, 初步结果表明MCM7能结合端粒酶催化亚基hTERT。为了阐明MCM7调控端粒酶活性的分子机制，我们拟开展以下几个方面的研究：（1）通过GST pulldown等方法确定MCM7与端粒酶hTERT的结合位点/结构域；（2）利用点突变和免疫共沉淀/免疫荧光等方法分析MCM7的磷酸化修饰是否影响其与hTERT的结合；探究MCM7是否影响hTERT与端粒酶RNA（hTR）的结合以及hTERT在细胞内的定位和稳定性；（3）建立MCM7及其功能位点突变体的表达细胞系，通过小鼠成瘤模型，分析MCM7是否通过上调hTERT活性而促进细胞成瘤性；（4）通过分析临床样本，明确MCM7蛋白水平与hTERT活性以及患者预后的相关性。

MCM （minchromosome maintenance）复合物在真核细胞的DNA复制起始过程中发挥关键作用。MCM复合体由MCM2-7组成，具有DNA解旋酶活性，在DNA复制起始过程中发挥重要作用。此外研究者发现当用siRNA把MCM7蛋白敲低时，DNA复制起始没有受到影响，这提示MCM7蛋白除了参与DNA复制起始以外还具有其它生物学功能。.本项目研究发现MCM7与端粒酶催化亚基hTERT存在相互作用，过表达MCM7能显著提高细胞内端粒酶活性；并发现MCM7存在于有活性的端粒酶复合物中。进一步研究发现MCM7与内源性端粒酶hTERT存在相互作用。为了探究MCM7调控端粒酶活性的分子机制，我们定位了MCM7与端粒酶hTERT的结合区域，发现MCM7与hTERT的结合不依赖于端粒酶RNA（hTR）。利用免疫共沉淀/免疫荧光等方法分析，发现MCM7影响hTERT蛋白的稳定性, 进一步研究发现MCM7可以通过抑制hTERT蛋白泛素化降解以及调节TERT的染色质结合水平来稳定TERT蛋白。我们还发现，MCM7 Ser121磷酸化是其上调端粒酶活性所必需的。此外TCGA数据分析进一步揭示了MCM7与TERT表达之间存在显著的正相关关系，且MCM7或TERT的表达与患者生存率呈负相关，提示MCM7和TERT的共高表达可能成为肿瘤患者诊断和预后的潜在标志物。总之，本研究发现MCM7能显著提高细胞内端粒酶活性，阐明MCM7与端粒酶催化亚基hTERT相互作用并提高其稳定性的分子机制，揭示MCM7除了参与DNA复制之外的新功能；本研究还提示MCM7和TERT有可能成为肿瘤患者诊断和预后的新型标志物。

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