采用ChIP-DSL启动子芯片筛选Kid下游调控基因

Identification of Kid Target Genes by ChIP-DSL Combined with Promoter Microarray

目的:前期研究发现驱动蛋白样DNA结合蛋白(kinesin-likeDNAbindingprotein,Kid)的编码基因(ki-nesin-like4,KNSL4)在乳腺原发癌中的表达下调与患者差的预后相关。本研究的目的是筛选Kid下游调控基因,为探讨Kid在乳腺癌发生和发展中的作用机制提供依据。方法:采用染色质免疫沉淀技术和含人类20045个功能基因启动子序列的Oligo芯片,筛选Kid抗体染色质免疫沉淀的DNA与芯片杂交的荧光强度,与无抗体对照比较差异达2倍以上的基因作为Kid调控的候选基因;采用ChIP-PCR方法验证候选基因的启动子与Kid的结合;采用实时定量RT-PCR方法验证Kid蛋白量改变对候选基因mRNA表达水平的影响。结果:筛选出287个Kid调控的候选靶基因;候选基因ATF7IP和CDC25C的Kid特异性染色质免疫沉淀扩增产物量与非特异对照比较差异分别为2.2倍和2.4倍;ATF7IP和CDC25C在KNSL4siRNA干扰的乳腺癌细胞中的表达水平较对照细胞分别下调1.6倍和5.8倍。结论:Kid对ATF7IP和CDC25C的转录呈负向调节作用;Kid可能通过对靶基因表达的转录调控进而影响乳腺癌的生物学行为。

Objective： Previous studies have shown that a downregulation of kinesin-like 4 （KNSLA） mRNA level is correlated with poor prognosis in breast cancer patients. The purpose of this study was to identify the target genes of kinesin-like DNA binding protein （Kid） anti to investigate the role of Kid in the carcinogenesis and development of breast cancer. Methods： DNA sequences occupied by Kid in MDA-MB-435S cells were profiled based on the chromatin immunoprecipitation- DNA selection and ligation （ChlP-DSL） method combined with promoter mieroarray experiments. The genes that were 2- fold or more differentially enriched by anti-Kid antibody compared to non-antibody were considered to be candidate genes. The candidate genes were validated by ChIP-PCR assay. The expression of candidate genes in MDA-MB-435S cells and MDA-MB-435S/KNSIA-siRNA cells was detected by real-time reverse transcription-PCR （RT-PCR） assay. Results： A total of 287 candidate genes were considered after ChlP-DSL and promoter microalTay. ChlP-PCR assay showed that ATF7IP and CDC25C genes were 2.2 and 2.4-fold enriched, respectively, by anti-Kid antibody. The expression of ATF7IP and CDC25C mRNA in MDA-MB-435S/KNSL4-siRNA cells was 1.6 and 5.8-fold upregulated compared with the parental cells. Conclusion： Kid negatively regulates transcription of the ATF7IP and CDC25C genes, and it may be involved in the carcinogenesis and development of breast cancer through this mechanism.