探测肿瘤转移抑制基因TMSG-1蛋白新功能：转录因子活性及转录调节功能

TMSG-1 was first identified as a new tumor metastasis suppressor gene by the Pathology Department of Health Science Center Pecking University in 1999. Our previous study for the first time found the truncated variants of TMSG-1 including Homeodomain could enter the nucleus.Then we analysed the protein sequence of TMSG-1 and we found it had nuclear localization signal (NLS) and DNA binding domain. Therefore, we for the first time proposed TMSG-1 could play the role of transcriptional factor after entering the nucleus. Our study aimes to investigate whether TMSG-1 could enter nucleus, as well as to detect its transcription regulatory function. The study comprises of the following aspects: ① Observing whether the inherent TMSG-1 in tumor cells could enter the nucleus under natural satus or certain stimulus. Detecting the truncated variants of TMSG-1 which could enter the nucleus using immunofluorescence and luser confocal microscopy after fused with His tag; ②According to the prediction of bioinformatics, carrying out site-specific mutation to identify the NLS of TMSG-1; ③Revealing the importin interacted with TMSG-1 by bioinformatics and immunoprecipitation to give insight into the mechanisms through which TMSG-1 enters the nucleus; ④Detecting the transcription regulatory function of TMSG-1 through luciferase report gene; ⑤Combining the chromatin immunoprecipitation and gene chip to screen and classify the down stream genes of TMSG-1 protein, as well as confirming the transcription regulatory function of TMSG-1 playing on the target genes through luciferase report gene, overexpression and RNA interfernce of TMSG-1.

TMSG-1是1999年北京大学医学部首先克隆出的肿瘤转移抑制基因。我们前期研究首次发现TMSG-1含Homeodomain结构域的截短体可以入核，进一步发现TMSG-1蛋白具有核定位信号及DNA结合功能域，因此我们在国内外首次提出TMSG-1蛋白可能入核并有转录因子活性。本课题重点研究肿瘤细胞TMSG-1入核及机制和转录调节作用。包括：①探测肿瘤细胞内TMSG-1蛋白自然状态或特定刺激下能否入核，TMSG-1与His融合进行免疫荧光及Westernblot检测其全长及截短体哪段可入核；②据生物信息学预测进行定点突变，确定TMSG-1核定位信号；③探讨TMSG-1入核机制，据生物信息学预测，免疫共沉淀探测与其相互作用的核输入蛋白；④荧光素酶报告基因检测TMSG-1是否具有转录调节功能；⑤染色质免疫共沉淀与基因芯片对其下游靶基因进行筛选，并通过荧光素酶、上调表达和RNA干扰验证转录调节作用。

TMSG-1是一个新的肿瘤转移抑制基因，前期研究发现其在体外抑制多种肿瘤细胞生长和侵袭。目前对于TMSG-1的研究主要是肿瘤转移抑制和神经酰胺合成方面。我们前期研究中发现TMSG-1部分区域能够入核，生物信息学分析发现TMSG-1蛋白含有homeodomain结构域和一个潜在核定位信号，因此我们首次提出，TMSG-1蛋白作为homeoprotein有可能具有转录因子活性发挥对基因表达的调节作用。.主要研究内容、重要结果及科学意义：.1、免疫荧光结果表明，在自然存在及刺激状态下，肿瘤细胞内均有TMSG-1蛋白表达，H2O2刺激下TMSG-1蛋白表达增加，且细胞核内蛋白表达增加，表明TMSG-1的表达和入核与应激反应相关；逆转录PCR及Western blot表明，H2O2刺激状态下TMSG-1基因及蛋白表达增加；.2、成功构建TMSG-1全长及不同截短体真核表达载体，免疫荧光表明，含有Homeodomain结构域的截短体T2、T3及T5能够入核，T1和不含Homeodomain结构域的T4定位于细胞浆。.3、构建核定位信号缺失体QST5真核表达载体，缺失体QST5不再定位于细胞核，主要位于细胞浆，表明homeodomain末尾119-128aa一段富含精氨酸的序列为其核定位信号序列，为进一步研究TMSG-1蛋白的转录功能奠定基础。.4、TMSG-1抗体免疫沉淀后，可以检测到Karyopherin a1、Karyopherin B1、Karyopherin B3和CRM1四种蛋白表达，说明TMSG-1蛋白可以和此四种核输入蛋白结合，这是TMSG-1蛋白可以入核的重要机制及证据。.5、染色质免疫共沉淀与高通量测序结合筛选TMSG-1蛋白下游靶基因共获得1978条基因片段信息，其中926条功能基因片段。进行GO与KEGG富集，其中多条基因与结肠癌、肝癌、肿瘤细胞坏死、RNA降解和神经元分化等功能相关。.6、选择下游靶基因DCC基因，双荧光素酶报告基因系统、上调和RNA干扰TMSG-1蛋白表达结果表明TMSG-1蛋白能够增强DCC基因的转录和表达，TMSG-1蛋白可以发挥转录因子作用调节DCC基因转录。.