C5orf21基因的分子克隆及在糖尿病大血管病变中的作用

目的 克隆与糖尿病大血管病变相关的新基因C5orf21基因,初步研究C50rf21基因在糖尿病大血管病变中的作用.方法 应用RT-PCR从人主动脉组织中克隆出C5orf21基因并经酶切、测序鉴定;研究C50rf21基因的组织表达谱、血管细胞表达谱以及高糖高脂对巨噬细胞中C5orf21基因表达的影响,并对C5orf21基因及蛋白功能进行分析.结果 成功克隆出CSorf21基因,人主动脉组织中存在5种C5orf21基因剪切体,编码框长度分别为1251、1113、894、810、810 bp,第3、5种剪切体GenBank数据库中尚未有收录,其中第4、第5种剪切体的蛋白编码框相同,均为810 bp,其差别主要在5′非翻译区碱基的不同,生物信息学分析发现C5orf21基因定位于染色体5q15,C5orf21含有Arb2结构域,可能与H3组蛋白第9位赖氨酸的甲基化有关;C5orf21组织表达较为广泛,以脂肪和主动脉组织表达最高;人主动脉内皮细胞、平滑肌细胞及巨噬细胞均有C5orf21基因的表达;高葡萄糖、油酸及LDL均上调巨噬细胞中C5orf21基因的表达.结论 首次在mRNA水平证实了C5orf21基因的存在,C5orf21基因表达的改变与糖尿病大血管病变有关.

Molecular cloning of a novel gene, C5orf21 gene and its roles in diabetic macroangiopathy

Objective To clone a novel diabetic angiopathy related protein gene- C5orf21 and study its roles in diabetic macroangiopathy. Methods The open reading frame (ORF) of C5orf21 gene was cloned into vector from human aortic tissues by a RT-PCR-based approach and identified by enzyme-cutting and sequencing. The structure and function of C5orf21 gene and protein were further analyzed by bioinformatics technology. The mRNA expression of C5orf21 gene in human tissues and in vascular cells was analyzed by RT-PCR. RT-PCR was used to observe the effect of high glucose, low-density lipoprotein (LDL) and free fatty acid (FFA) upon the expression of C5orf21 gene in macrophnges. Results C5orf21 gene was successfully inserted into plh-ive vector and identified for the first time at the level of mRNA. There are five C5orf21 gene splice variants in human aortic tissue and their length of ORF are 1251, 1113, 894, 810 and 810 bp respectively. Two kinds of splice variants have yet to be included in GenBank database. Two kinds of splice variants have the same ORF and their differences are mainly in the bases in the 5' untranslated region. Bioinformatics analysis found that C5orf21 gene was located in chromosome 5q15 and C5orf21 protein contained Arb2 domain associated with histone H3 lysine 9 methylation. C5orf21 gene was normally expressed in many tissues. Fat and aortic tissues had the highest expression. The expression of C5orf21 gene could be detected in human aortic endothelial cell, aortic smooth muscle cell and macrophages. High glucose, LDL and FFA (esp. high glucose) up-regulated the expression of C5orf21 gene in macrophage. Conclusion C5orf21 gene contains five splice variants and it is identified for the first time at the level of mRNA. The changes of C5orf21 gene expression are correlated with diabetic macroangiopathy.