Identification of targetgenes of E1AF using two hybrid system

E1AF是ets-癌基因家族转录因子的一员，E1AF被证明与MMPs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Ad12型E1A (Ad12E1A)，一种高致瘤性腺病毒产物能够抑制癌细胞系的侵袭能力。用Ad12E1A表达质粒转染高侵袭性口腔鳞状细胞癌HSC3，建立12-12S细胞系。与亲代HSC3细胞相比，12-12S细胞中E1AF mRNA的表达降低。MMPs在12-12S细胞中mRNA水平和蛋白水平均有所降低。此外，Matrigel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，进而通过下调MMP表达抑制人类癌细胞的侵袭能力。这可能表明ad12e1a和E1AF之间存在负反馈电路。我们检测了HGF对HSC3侵袭性潜能方面的E1AF和MMP基因表达的影响。HGF刺激E1AF基因的表达。在HGF处理的细胞中，MMP-1、-3和-9 mrna水平升高，并与E1AF上调相关。反义E1AF表达载体转染亲代HSC3细胞后，ASE1AFHSC3细胞中MMP-1和MMP-9 mRNA表达未见明显上调。这些结果表明，HGF诱导ets相关的E1AF转录因子基因的表达，其产物反过来激活MMP基因，导致口腔癌细胞侵袭。

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.