人类TBC1D8B基因两个转录本的功能研究

发布时间：2018-06-26 17:36

本文选题：人特异基因 + 可选择性多聚腺苷酸化　；参考：《云南大学》2016年博士论文

【摘要】：TBC结构域蛋白家族是含有一段由保守的氨基酸构成的TBC结构域的GAP蛋白(TBC/RABGAPs),可以特异识别并调节Rab活化状态,该家族蛋白的生物学功能与细胞膜紧密相关。因此,TBC/RABGAPs在调节细胞信号传导和细胞内其他的活动方面发挥重要作用,特别是调控肿瘤发生发展方面起着重要作用。可选择性多聚腺苷酸化(alternative polyadenylation, APA)普遍存在于真核生物中。由于APA位点的存在,基因可以通过不同的剪切引起编码区和3'UTR的改变,从而产生不同的转录产物。因此,APA也可以认为是新基因产生的一种机制。通过前期的比较基因组学分析,我们发现在人类TBC1D8B基因可以通过APA机制产生两种转录本(TBC1D8B-a和-b转录本),TBC1D8B-a在哺乳动物中广泛存在,TBC1D8B-b是人特异的。但是TBC1D8B的两个转录本的生物学功能尚不清楚。首先,我们筛选出适用于qPCR分析中最适内参基因ACTB,应用于后续的基因相对表达量研究；接下来,我们对TBC1D8B-a和-b转录本的功能开展系统地研究,结果显示：(1)由插入序列产生的TBC1D8B-b的3'UTR的序列无论是在不同人群,还是在不同的疾病组中,这段序列无任何突变位点,是高度保守的,提示可能具有重要的生物学功能；(2)实验验证了TBC1D8B-b以及编码的蛋白Tbc1d8b-b是人类特有的；(3) TBC1D8B基因的两个转录本在人类大多数正常组织或细胞中均有表达,而且TBC1D8B-a基因在前列腺癌、结直肠癌、肺癌中的表达显著高于正常组织；(4) TBC1D8B-a显著促进细胞增殖和DNA合成,加速细胞周期进程,提高细胞迁移和浸润能力,具有原癌基因的特征；当TBC1D8B-a活性中心位点突变(Arg538Lys和Gln573Leu)后,上述的功能全部丧失,表明TBC1D8B-a基因的功能依赖于其TBC domain的活性；(5)野生型TBC1D8B-b对细胞增殖、细胞周期进程和细胞迁移无明显影响,但是,当TBC domain的活性位点突变(Arg538Lys和Gln573Leu)后,会显著促进细胞增殖、细胞周期进程和细胞迁移；(6)利用高通量测序技术,我们分析了过表达TBC1D8B-a-wt, TBC1D8B-a-mt, TBC1D8B-b-wt和TBC1D8B-b-mt细胞株的转录组,预测了野生型TBC1D8B-a和突变型TB1C1DB-b可能的信号通路,发现两个转录本可能参与不同的信号通路。本研究是首次对TBC1D8B基因两个转录本开展的功能研究,不仅丰富了我们对TBC结构域蛋白家族的认识,而且增加了对人特异基因的起源及功能的认识,可能为癌症或疾病的诊断和治疗提供新的靶点,进一步可能为癌症或疾病的预防提供新的思路。
[Abstract]:The TBC domain protein family is a gap protein (TBC / RABGAPs) consisting of a conserved amino acid domain, which can specifically recognize and regulate the activation of Rab. The biological function of TBC domain protein is closely related to the cell membrane. Therefore, TBC / RABGAPs play an important role in regulating cell signal transduction and other cellular activities, especially in the regulation of tumorigenesis and development. Selective polyadenosylated (alternative polyadenylation, (APA) is commonly found in eukaryotes. Because of the existence of APA sites, genes can produce different transcriptional products by changing the coding region and 3UTR through different splicing. Therefore, APA may also be considered as a mechanism for the production of new genes. By previous comparative genomics analysis, we found that human TBC1D8B gene can produce two kinds of transcripts (TBC1D8B-a and -b transcripts) through the APA mechanism. TBC1D8B-a is widely present in mammals and TBC1D8B-b is human specific. However, the biological functions of the two transcripts of TBC 1 D 8 B are unclear. First, we screened the most suitable internal reference gene ACTB for qPCR analysis, and applied it to the subsequent studies of relative gene expression. Then, we systematically studied the function of TBC1D8B-a and -b transcripts. The results showed that: (1) the 3UTR sequence of TBC1D8B-b produced by the insertion sequence was highly conserved, no matter in different population or in different disease groups, indicating that it may have important biological function; (2) TBC1D8B-b and the encoded protein Tbc1d8b-b are unique to human beings. (3) the two transcripts of TBC1D8B gene are expressed in most human normal tissues or cells, and TBC1D8B-a gene is expressed in prostate cancer and colorectal cancer. (4) TBC1D8B-a significantly promoted cell proliferation and DNA synthesis, accelerated cell cycle progression, enhanced cell migration and infiltration, and had the characteristics of proto-oncogene, after TBC1D8B-a mutation (Arg538Lys and Gln573Leu). All the above functions were lost, indicating that the function of TBC1D8B-a gene depended on the activity of TBC domain. (5) the wild-type TBC1D8B-b gene had no significant effect on cell proliferation, cell cycle progression and cell migration. However, when the active site mutation of TBC domain (Arg538Lys and Gln573Leu) was observed, TBC1D8B-b had no significant effect on cell proliferation, cell cycle progression and cell migration. (6) by using high-throughput sequencing, we analyzed the transcriptional pathways of overexpression of TBC1D8B-a-mt, TBC1D8B-b-wt and TBC1D8B-b-mt cells, and predicted the possible signaling pathways of wild-type TBC1D8B-a and mutant TB1C1DB-b. Two transcripts may be involved in different signal pathways. This is the first functional study of two transcripts of TBC1D8B, which not only enriches our understanding of the TBC domain protein family, but also increases our understanding of the origin and function of human specific genes. It may provide new targets for the diagnosis and treatment of cancer or disease, and further provide new ideas for the prevention of cancer or disease.
【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R394

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