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Identification of targetgenes of E1AF using two hybrid system

使用两种混合系统鉴定E1AF的靶基因

基本信息

批准号：
10671694
负责人：
SHINDO Masanobu
金额：
$ 2.11万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671694/
关键词：
E1AF Ad12E1A HGF DNA microarray etsがん遺伝子群転写因子結合タンパク EIAF ets がん遺伝子 yeast two hybrid RNAサブトラクション法

项目摘要

E1AF is a member of the ets-oncogene family transcription factor, and E1AF was shown to bind to the promoter region of MMPs that induce transcription from multiple MMP genes.In the present study, we demonstrated that Ad type 12 E1A (Ad12E1A), a highly tumorgenic adenovirus product is able to suppress the invasion ability of a cancer cell line.12-12S cell lines were established by transfection with Ad12E1A expression plasmid into HSC3, a highly invasive oral squamous cell carcinoma cell line. Expression of E1AF mRNA decreased in 12-12S cells compared to that of the parental HSC3 cells. Reduced expression of MMPs in 12-12S cells was identified both in mRNA levels and in protein levels. Furthermore, in vitro invasion assay using Matrigel revealed that the invasion ability of 12-12S cells was suppressed by half compared to that of HSC3 cells.These results suggest that Ad12E1A downregulates E1AF which, in turn, restrains invasion ability of human cancer cells by way of downregulaton of MMP expression. This may suggest that a negative-feedback circuit exists between Ad 12 E1A and E1AF.We examined the effect of HGF on the E1AF and MMP gene expression in terms of the invasive potential of the HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and MMP-9 mRNA was observed in ASE1AFHSC3 cells that was transfected with antisense E1AF expression vector into parental HSC3 cells.These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.

E1AF是ets-oncogene家族转录因子的成员，E1AF被证明可以与MMP的启动子区域结合，诱导多个MMP基因的转录。在本研究中，我们证明了Ad type 12 E1A（Ad12E1A）这种高致瘤性腺病毒产物能够抑制癌细胞系的侵袭能力。通过转染建立12-12S细胞系 Ad12E1A 表达质粒进入 HSC3（一种高度侵袭性口腔鳞状细胞癌细胞系）。与亲本 HSC3 细胞相比，12-12S 细胞中 E1AF mRNA 的表达降低。 12-12S 细胞中 MMP 的 mRNA 水平和蛋白质水平表达均减少。此外，使用Matrigel进行的体外侵袭实验显示，与HSC3细胞相比，12-12S细胞的侵袭能力被抑制了一半。这些结果表明，Ad12E1A下调E1AF，而E1AF反过来又通过下调MMP表达来抑制人类癌细胞的侵袭能力。这可能表明Ad 12 E1A和E1AF之间存在负反馈回路。我们从HSC3的侵袭潜力方面检查了HGF对E1AF和MMP基因表达的影响。 HGF 刺激 E1AF 基因的表达。用 HGF 处理的细胞中 MMP-1、-3 和 -9 mRNA 的水平增加，并且与 E1AF 上调相关。相比之下，用反义E1AF表达载体转染亲本HSC3细胞的ASE1AFHSC3细胞中，没有观察到MMP-1和MMP-9 mRNA的明显上调。这些结果表明，HGF诱导Ets相关E1AF转录因子基因的表达，其产物反过来激活MMP基因并导致口腔癌细胞侵袭。