【摘要】 目的:利用生物芯片技术分析6种不同器官肿瘤细胞中microRNA(miRNA)的表达差异。方法:将210个与已知人类和小鼠miRNA互补的序列(206个miRNA,4个阳性对照)作为探针,点于玻片上制备寡核苷酸芯片。提取肿瘤细胞HeLa(人宫颈癌上皮细胞)、MCF-7(人乳腺癌细胞)、A549(人肺腺癌细胞)、HT-29(人结肠腺癌细胞)、ES-2(人卵巢透明细胞癌)、K562(人慢性髓细胞性白血病细胞)的miRNA,荧光染料Cy3标记,并与制备好的芯片杂交;用ScanArrayTM Express1.0扫描仪扫描荧光信号,采用ScanArray3.0和Cluster3.0软件分析处理扫描结果;Northern blotting和RT-PCR方法对芯片检测结果进行验证。结果:在6种不同器官肿瘤细胞中,检测到115种miRNA存在差异,91种miRNA没有明显差异;其中,miR-21在6种肿瘤细胞表达水平均较高,miR-125b表达水平均较低,let-7不同亚型在6种细胞系中表达水平较低;miR-17-5p和miR-20a呈集簇表达,在卵巢肿瘤ES-2细胞中的表达水平高于其他细胞,乳腺肿瘤细胞系MCF-7和宫颈癌细胞系HeLa的miRNA表达谱聚为一类。Northern blotting检测到miR-17-5p及其前体在K562细胞中均见表达,A549和ES-2细胞中只见较弱的前体表达,MCF-7、HeLa和HT-29中可见明显的前体和较弱的成熟miRNA的表达。RT-PCR检测到miR-17-5p前体在K562细胞中表达水平高于其他几种细胞,在ES-2细胞中的表达量低于K562细胞,同时高于另外4种细胞;miR-21在6种肿瘤细胞中表达水平均较高,在A549细胞中表达最高。结论:应用生物芯片技术检测肿瘤细胞中miRNA的表达差异为进一步探索miRNA与肿瘤间的关系奠定基础。更多还原

【Abstract】 Objective:To determine the difference of miRNA expression profiles in 6 types of human cancer cells by microarray technique.Methods:The microarray was prepared,with contained 210 oligonucleotides,including 206 probes complementary with human and mouse miRNA sequences and 4 positive control oligos.MiRNAs were extracted from HeLa(human cervical cancer epithelial cells),MCF-7(human breast cancer cells),A549(human lung adenocarcinoma cells),HT-29(human colonic cancer cells),ES-2(ovarian carcinoma cells),and K562(chronic myelogenous leukemia cells) cells and were labeled with Cy3 for hybridization to the miRNA microarray.The slides were scanned by ScanArrayTMExpress1.0 and images were analyzed by ScanArray3.0 and Cluster3.0;the results were confirmed by Northern blotting and RT-PCR.Results:Totally 115 miRNAs were found to be differentially expressed in the 6 cancer cell lines,with miR-21 expression up-regulated and miR-125b,let-7 expression down-regulated.The expression of miR-17-5p and miR-20a was in cluster and was more higher in ES-2 cells than in other cells.HeLa and MCF-7 cells were located on a single branch of the dendrogram in cluster analysis.Northern blotting showed that both pre-and miR-17-5p expressed in K562,pre-miR-17-5p was weakly expressed in A549 and ES-2 cells,and obvious pre-miRNA expression and weak miRNA expression were noticed in MCF-7,HeLa,and HT-29 cells.RT-PCR showed that expression of pre-miR-17-5p in K562 cells was higher than those in other cells.Expression of miR-21was high in all 6 cell lines and the highest expression was seen in A549 cells.Conclusion:Microarray can be used to detect miRNA expression files in cancer cells,which contributes to the study of the relation between miRNA and tumor.更多还原