转录因子E2F1抑制CREG表达调控体外培养的人血管平滑肌细胞分化  

Transcriptional Factor E2F1 Inhibits Expression of The Cellular Repressor of E1A-Stimulated Genes and Regulates VSMCs Differentiation In vitro

作　　者：韩雅玲[1] 赵昕[1] 闫承慧[1] 康建[1] 张效林[1] 邓捷[1] 徐红梅[1] 刘海伟[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科,沈阳110016

出　　处：《生物化学与生物物理进展》2007年第5期490-496,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(30570664);辽宁省自然科学基金(2050426)资助项目~~

摘　　要：为探讨转录因子E2F1在血管平滑肌细胞(vascular smooth muscle cells,VSMCs)表型转化中的作用及其对E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)表达调控的分子机制,应用生物信息学方法,定位人CREG(hCREG)基因启动子并确定转录因子E2F1在hCREG启动子区的结合位点,PCR方法克隆并构建hCREG基因启动子绿色荧光报告基因载体,以hCREG启动子区E2F1结合位点为模板,化学合成E2F1寡聚脱氧核苷酸(ODN)和错配E2F1ODN,利用转录因子"诱骗(Decoy)"策略,用E2F1ODN转染体外培养的VSMCs以阻断E2F1与hCREG基因启动子区的结合,蛋白质印迹(Western blot)分析检测阻断前后细胞内hCREG蛋白、报告基因绿色荧光蛋白(green fluorescent protein,GFP)和平滑肌细胞分化标志蛋白SMα-actin表达变化.结果显示:分化表型HITASY细胞中E2F1表达下调伴出核转位,而增殖表型的HITASY细胞中E2F1蛋白表达明显增加且定位于核内.进一步应用FuGene6瞬时转染E2F1ODN和错配E2F1ODN于体外培养HITASY细胞中,蛋白质印迹分析发现,转染E2F1ODN后,HITASY细胞中hCREG、SMα-actin和GFP表达均较未阻断组及错配组细胞明显增加.上述研究结果证实,E2F1是hCREG基因转录的重要调控因子,能够直接结合于hCREG启动子区阻遏hCREG表达,参与hCREG蛋白对VSMCs表型转化的调控作用.The human cellular repressor of E1A-stimulated genes （hCREG）, originally cloned from the cDNA library of HeLa cell line, was found to rapidly induce the differentiation of diverse type cells such as pluripotent mouse EC cells, monocytes U937 and myeloid cells EML. It was identified in previous study that not only could overexpression ofhCREG regulate and hold the differentiation vascular smooth muscle cells （VSMCs） in vitro, but inhibit the neointima formation in rat carotid artery after balloon injury. These properties suggest that hCREG might have played an important role in antagonizing restenosis of vessel by inhibiting phenotypic modulation of VSMCs. In order to further elucidate the biological functions of hCREG in VSMCs, the upstream molecule mechanisms in regulating its expression were analyzed. At first, bioinformatics was used to predict the hCREG promoter and the binding sites of transcriptional factors. According to bioinformatic results, the pEGFP-hCREG-P945 reporter gene vector was constructed successfully which contained upstream 945 bp of hCREG where two binding sites of E2F1 were determined. Subsequently, the hCREG promoter activity was identified directly by detecting the expression of reporter protein-GFP with fluorescence microscope and Western blot analysis when the vector was transfected into human VSMCs-HITASY. Secondly, both the expression of hCREG and smooth muscle α-actin（SM α-actin） was detected to increase in differentiation HITASY cells cultured for 72 h with serum deprivation, in which the expression of E2F1 was reduced significantly. Inversely, the increase of E2F1 expression was detected in dedifferentiation cells cultured with 10% FBS medium accompanied with the reduction ofhCREG and SM α-actin. It is suggested that E2F1 maybe inhibit the expression ofhCREG by binding to the hCREG promoter. Furthermore, the E2F1 oligodeoxynucleotide （ODN） and mismatch E2F10DN were designed and used to block the binding of E2F1 to hCREG promoter by transcriptional factor ＂decoy

关 键 词：E2F1 转录因子 阻遏蛋白 E1A 表型 血管平滑肌细胞 

分 类 号：Q463[生物学—生理学]