应用基因芯片研究As_2O_3诱导K562细胞前后差异表达基因

Investigation of the differential expression genes of K562 cells induced by arsenic trioxide with microarray

目的研究三氧化二砷(As2O3)处理K562细胞前后,基因表达的变化。方法提取As2O3作用K562细胞前后的总RNA,逆转录成cDNA并用Cy3标记对照组,Cy5标记处理组,与自制的包含162个cDNA片段的K562芯片杂交。结果K562细胞经As2O3作用后,162个基因片段的表达都有所降低,其中25个基因片段的表达降低较为显著(Cy5/Cy3<0.5)。这些表达下调的基因片段包括转导因子、代谢酶、信号通路蛋白、激酶等,有6个差异表达基因与细胞的凋亡通路密切相关。As2O3可能通过抑制胰岛素样生长因子的功能,诱导细胞凋亡。结论应用自制的基因芯片筛选到了As2O3诱导K562细胞前后差异表达的基因,主要与细胞凋亡密切相关。

Objective To identify the difference in gene expression profile of K562 cells induced by arsenic trioxide. Methods The probes about 200 bp -500 bp derived from PCR-amplified products were spotted on the microscope slides to produce 30 × 18 microarrays.Total RNA was extracted by using Trizol reagents, 40μg of total RNA labeled by reverse transcription using Superscript Ⅱ transcriptase, oligo （dT） primer, and Cy5-dCTP or Cy3-dCTP for experimental and control samples, respectively. Then the labeled cDNAs were hybridized to microarrays at 42 ℃ for 12 - 18 h. The microarrays were scanned by using a dual-laser scanner and the experimental data were analyzed with the Quantarray software package. Results Among the 540 spotted microarray, 25 genes were found down-regulated, including transcription factors, metabolic emzymes, signal transduction proteins and kinases etc. Further analysis revealed about 6 genes were involved in the apoptosis pathway. Moreover, we suggested that the apoptosis induced by arsenic trioxide was the results of inhibiting the functions of IGFs. Conclusion Our results demonstrated that the cDNA mieroarray contracted by us could successfully apply to analysis the expression profile of hundreds of genes and screen the differential expression genes.