中文摘要：

缺氧诱导丝裂原因子（hypoxia-induced mitogenic factor,HIMF）是一种肺组织特异性生长因子,可刺激肺细胞增生和再生,但HIMF基因表达调控的机制尚未明确.为探索HIMF基因转录调控机制,首先以小鼠基因组DNA为模板,通过PCR扩增方法获得-348-＋61bp、-302-＋61bp、-131-＋61bp、-68-＋61bp的HIMF启动子片段,再将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子ETS-1结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染正常氧浓度条件下培养的小鼠肺上皮MLE-12和MLE-15细胞、结肠癌CT26细胞.结果发现,各HIMF启动子片段在MLE-12、MLE-15细胞中均有活性,但在CT26细胞中活性缺失;-302--131bp区存在HIMF启动子的核心调控元件.针对该区域ETS-1转录因子结合位点进行突变或缺失,能导致HIMF启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合ETS-1.结果提示,转录因子ETS-1参与正常氧浓度下HIMF启动子活性的调控,为研究HIMF基因的转录调控机制奠定了实验基础.

英文摘要：

Hypoxia-induced mitogenic factor （HIMF） is a lung-specific cytokine, which plays critical roles in the proliferation and regeneration of lung epithelial cells. However, the regulation of HIMF gene expression remained unclear. To explore the mechanisms of HIMF gene transcription, four segments of HIMF promoter, -348- ＋61 bp, -302 - ＋ 61 bp, -131 - ＋ 61 bp, and -68 - ＋ 61 bp, were obtained by PCR amplification from mouse genomic DNA and then subcloned into pGL3-Basic vectors containing a luciferase reporter. The mutation or deletion constructs at the binding site of transcription factor ETS-1 within the HIMF promoter were also prepared. The recombinant reporter vectors were transiently transfected into mouse lung epithelial cell lines MLE-12 and MLE-15, as well as colon cancer CT26 cells by Lipofectamine 2 000 under nomoxia. The results have shown that all the HIMF promoters were activated in MLE-12 and MLE-15 cells, but not in CT26 cells. The - 302 - - 131 bp region was determined to be the core element of the HIMF promoter, containing a ETS-1 binding site as shown by EMSA. The mutation or deletion within this region resulted in a significant decrease of the promoter activity. Our results suggested that ETS-1 played an important role in the activation of HIMF promoter under nomoxia. The discovery may help further studies of the regulatory mechanisms of the HIMF gene transcription.