中文摘要：

课题组前期工作表明GADD45β和c-Fos在巴西苏木素（Brazilin）致死膀胱癌细胞T24时显著升高,且分别过表达GADD45β和c-Fos均能抑制T24细胞的生长,表明其为Brazilin致死T24细胞潜在的靶标基因,但并未在GADD45β和c-Fos沉默状态下研究Brazilin对T24细胞的影响。RNA干扰是目前沉默靶标基因的常用技术手段,但高效RNAi片段筛选步骤繁琐,成本高。在本文中,首先以GADD45β为例,设计仅需一步酶切连接就可获得的荧光报告载体p-GADD45β-EGFP-N1,对GADD45β的3条siRNA（siRNA360、siRNA477和siRNA638）在荧光显微镜下进行干扰效率的检测,获得的干扰效率分别为63%、34%和67%,表明siRNA638对GADD45β干扰效率最高,与RT-qPCR的筛选结果一致。为验证荧光报告载体的通用性,选择c-Fos构建荧光报告载体p-c-Fos-EGFPN1,通过筛选获得荧光量最少的c-Fos siRNA274组的干扰效率最高为64%,与RT-qPCR的验证结果一致。

英文摘要：

In our previous work,it was demonstrated that GADD45fl and c-Fos were increased significantly while the bladder cancer T24 cells were treated by Brazilin. Meanwhile,the overexpression of GADD45fl and c-Fos could also inhibit the growth of T24 cells, and the results indicated that both GADD45fl and c- Fos were the potential target genes involved in killing T24 cells by Brazilin. So it is necessary to research the influence of Brazilin on T24 cells under the deficiency of GADD45fl and c-Fos to confirm the crucial roles of these two genes. RNA interference （RNAi） is an important tool and widely used for gene function research in human cancer cells. However, screening the efficient RNAi fragments is a tedious and time-con- suming job. In this study, we first constructed the fluorescence reporter vector p-GADD4513-EGFP-N1 which contained the co-expression cassette GADD45fl and GFP, then the silencing efficiency of siRNAs for GADD45fl in T24 cells was detected through the GFP florescence signals. Using this technology, we foundthat the interference efficiencies of GADD45fl siRNA360, GADD45fl siRNA477 and GADD45fl siRNA638 were 63%, 34% and 67%, which were compatible with results of RT-qPCR, and the best one was siR- NA477. To further validate the generality of the screening system, we constructed the other fluorescence reporter p-c-Fos-EGFP-N1 and obtained c-Fos siRNA274 with the highest 64% inhibition efficacy, which could also match with the results of the RT-qPCR.