Menin调控mRNA转运与内分泌腺体肿瘤发生的作用机制研究

The MEN1 gene has been reported to be responsible for the MEN1 (Multiple endocrine neoplasia type I), but its molecular function as well as the exclusive susceptibility of its mutations to endocrine glands remains a mystery even to this day. Preliminary data from our lab verified that Menin, encoded by MEN1, can regulate the energy metabolism-related genes, such as CRTC1, G6pase and PKA, with a pattern like a shock absorber: augment the weak signal and curb the strong signal, which suggests that Menin can control the signal amplitude of growth hormone-G protein coupling receptor-PKA-cAMP signal path. We also found that Menin was involved in the mRNA processing and transporting path, which was evidenced by the interaction of Menin with THO-TREX complex members and its co-immunoprecipitation with CRTC1 and G6pase mRNA. In this project, we will first set up the monoallelic and double ablation of MEN1 gene in pancrea β cell lines, and then to check our point in this unique model that Menin can protect the endocrine cells themselves from stress within such an environment with very high and volatile concentration of growth hormone. Secondly, we will attempt to find how Menin interacts and regulates the THO-TREX complex to mediate the hormone signal and mRNA processing/transporting process. Finally, we will further verify our story of Menin in mono-allele or biallele knockout mouse models. From this project, the genotype and phenotype of MEN1 gene versus MEN1 syndrome will get a more reasonable correlation and possibly helps to find clues of ways to cure this disease clinically.

MEN1基因突变引发多内分泌腺体肿瘤，目前该基因的功能及其突变特异引发多种内分泌腺体肿瘤的机制依然不明确。我们的初步研究结果发现，该基因所编码蛋白Menin能够以一种奇特的"增小抑大"的模式调控能量代谢基因CRTC1、G6pase的表达和PKA激活剂的作用强度，类似减震器的功能；研究结果也证明Menin与THO-TREX复合物互作，该复合物参与mRNA转录、加工、组装和转运。本项目通过构建MEN1单等位和双等位敲除的内分泌细胞模型，验证Menin调控的模式是通过调控内分泌腺体细胞对自身分泌的高浓度激素的耐受能力，防止因激素浓度剧烈变化而引发自身信号过度波动；验证Menin是通过调控mRNA加工/转运环节（THO-TREX复合物）来调控基因的表达；并且通过建立MEN1单等位和双等位敲除小鼠模型，进一步在体验证Menin的生物功能。本项目研究将为阐明内分泌腺体肿瘤发生的作用机制提供理论依据。

在临床上，MEN1基因突变引起较广泛的内分泌腺体肿瘤，而非内分泌腺体细胞则很少发生肿瘤，对此国际上依然未有合理的解释，原因在于缺乏对该基因功能的深入认识；目前报道的MEN1的蛋白Menin能够与几十种蛋白互作，但目前没有任何一个蛋白的互作能够解释其引发内分泌腺体肿瘤的机制问题。针对此问题，在内分泌细胞和非内分泌细胞中，我们使用RNAi干扰MEN1后比较了其对代谢、细胞周期和PKA信号通路等的影响。结果显示非内分泌细胞和内分泌细胞在MEN1表达降低后，细胞周期、代谢、PKA信号通路的表达变化相似，而且较强的干扰之后，均出现细胞周期阻滞和凋亡的现象，低强度敲低MEN1后均出现细胞生长加快的现象，但是在insulin和forskolin等刺激2种来源的MEN1敲低细胞后，内分泌细胞和非内分泌细胞间并未出现明显差别，说明MEN1对内分泌和非内分泌源的细胞的作用机制类似；由此我们推测MEN1突变后对不同细胞的影响差异可能与细胞所处的激素环境有关，进一步的实验结果显示，MEN1过表达后对CRTC1（增强PKA信号）具有增小抑大的作用，低水平的过表达CRTC1后，MEN1增强CRTC1的表达，高水平表达CRTC1，MEN1反而增强了对其表达水平的抑制和对creb1 S133磷酸化水平的抑制，减弱CRTC1对含有cre元件启动子的启动增强作用；不仅如此，MEN1和CRTC1的共同作用能够改变PKAcpT198入核，改变其核质比分布；另外改变了NUP62等核孔复合物成分和THOC5等核输出成分的胞质和胞核比例，MEN1倾向于增加NUP62和THOC5在细胞核中的分布，CRTC1倾向于降低这些成分在细胞核中的分布，二者共同过表达后倾向于大大增加在胞质和胞核中的分布；在Cre_ERT2-tamoxifen介导的MEN1条件性敲除小鼠实验中也显示，tamoxifen处理后的纯合小鼠中的黑色素细胞增生，黑色素沉着增加。该结果首次证实了MEN1与外部环境中的激素之间的调控关系，防止PKA信号激素变化波动给细胞带来的过大的代谢波动，这可能是MEN1在一些内分泌腺体中的关键作用；首次证明了MEN1在调控核输出和输入环节中的作用，大大增进对其功能和所引发肿瘤的分子机制的认识。