促细胞增殖新基因TOX的功能研究

摘要

The completion of human genome project has led to the identification of thousands of novel genes,most of which remain unknown or poorly understood functions.Cell proliferation is a highly ordered operation process,and it plays an important role in the life activities.The abnormal regulation of cell proliferation often causes a variety of disease occurrence and development.So,the identification and function study of human functional genes involved in regulation of cell proliferation will provide new directions for mechanism research of cell proliferation and therapy strategy of various related diseases.By searching the human RefSeq and expressed sequence tag (EST) databases in GenBank,human function gene TOX (thymocyte selection-associated high mobility group box) (NCBI RefSeq No:NM_014729) was cloned from human multi-tissue cDNA library by using PCR (polymerase chain reaction) assay.Bioinformatics analysis was used to identify structural characteristics of TOX,and RT-PCR (reverse transcription PCR) was used to detect its expression in cell lines.Fluorescence microscopy was used to analyze subcellular localization of TOX,moreover,cell growth curve assay and flow cytometry experiment were used to study its effect on cell proliferation and cell cycle.The results showed that the full length human gene TOX was 4 131 bp long with an in frame stop codon upstream of the putative ATG start codon and a 3'-poly(A) tail,and the open reading frame (ORF) encoded 526 amino acids with a predicted molecular mass of 57 ku.The human gene TOX was located on chromosome 8q12.1,and encompassed nine exons and eight introns.Bioinformatics analysis showed the human gene TOX might locate in cell nuclei.TOX-coding protein was one of high mobility group box family members,which were involved in gene regulation of physiological processes.RT-PCR analysis revealed that TOX was highly expressed in leukemia cell lines Jurkat and Raji,but was not detected in some tumor cell lines A549,H1299,H520.The analysis by constructing a TOX-GFP fusion plasmid showed that the TOX-GFP could co localize with DAPI,a nucleus-specific fluorescent dye,suggesting that the subcellular localization of TOX was in the nucleus.There was a noted increase in cell proliferation during a 5-day period by CCK8 assay,and the overexpressed TOX could accelerate the cell growth rate,and increase the ratio of cells in S phase by flow cytometry analysis.In conclusion,human gene TOX encodes a HMG-box family protein,which can regulate gene expression such as transcription and replication through binding with DNA.TOX may regulate some gene expression in cell cycles and it is highly expressed in T and B lymphoma cell lines,suggesting that the TOX gene may regulate the development of diseases related to T and B cells.Although TOX particular mechanism needs further research,the present results indicate that the human gene TOX is involved in the regulation of cell proliferation,and might be developed as a drug target or disease marker.%利用GenBank中的数据,通过聚合酶链式反应(polymerase chain reaction,PCR)从人的cDNA文库中克隆得到功能基因TOX(thymocyte selection-associated high mobility group box),运用生物信息学分析其结构特性,并通过逆转录PCR(reverse transcription PCR,RT-PCR)分析TOX在细胞系中的表达；利用荧光显微镜观察其亚细胞定位情况；使用细胞生长曲线实验和流式细胞术分析TOX对细胞增殖及细胞周期的影响.结果表明:TOX基因定位于人8号染色体q12.1上,全长4 131 bp,编码526个氨基酸,该蛋白质分子质量约为57 ku,合有9个外显子和8个内含子,其编码蛋白为高迁移率蛋白家族成员,能参与基因调控等生理过程；TOX基因在白血病细胞系Jurkat和Raji中高表达,并定位于细胞核中；对细胞生长曲线绘制及细胞周期分析表明,TOX能够使细胞生长速度加快,且S期细胞比例明显上升.说明TOX参与了细胞增殖调控,有可能研究开发为药物靶标或疾病标志物.