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目的:结合生物信息学分析手段,筛选乳腺癌转移相关微RNA(microRNA,miRNA)-200c调控的基因网络。方法:采用Aymetrix miRNA生物芯片分析12株乳腺细胞中差异表达的miRNA;应用脂质体转染法将miR-200c模拟物(mimic)转入4株高转移性的乳腺癌细胞株(BT549、HS578T、MDA-MB-231和SUM159PT)中,再用Aymetrix mRNA生物芯片检测转染miR-200c mimic后高转移性乳腺癌细胞株中差异表达的基因。采用生物分子功能注释系统(Capital Bio Molecule Annotation System,MAS),筛选miR-200c调控的信号通路与基因网络。结果:12个乳腺细胞株中筛选出9个差异表达的miRNA(P<0.01,倍数值≥20或≤-20),其中以miR-200c在高转移性乳腺癌细胞株中下调幅度最显著。4个转染miR-200c mimic的乳腺癌细胞株中共有33个共上调基因及13个共下调基因。实时荧光定量-PCR与蛋白质印迹法验证结果显示,共调基因锌指E框结合同源框1(zinc finger E-box binding homeobox1,ZEB1)mRNA和蛋白在4个转染细胞株中均下调。MAS生物信息学分析结果显示,转染miR-200c mimic的乳腺癌细胞株中共有的差异表达基因相关信号通路包括嗅觉传导(olfactory transduction)通路、细胞因子-细胞因子受体关联(cytokine-cytokine receptor interaction)通路和细胞黏附分子(cell adhesion molecules,CAMs)通路等,不同的信号通路可以通过共调基因相互联系,在特定的信号通路中,共调基因间也存在密切的相互联系。结论:以高通量生物芯片检测为基础,运用生物信息学分析手段,多元化筛选获得miR-200c调控的基因网络,为后续展开miR-200c作用机制的研究提供了明确的方向。
OBJECTIVE: To screen a gene network regulated by microRNA (miRNA) -200c in breast cancer combined with bioinformatics analysis. Methods: The miRNAs differentially expressed in 12 breast cells were analyzed by Aymetrix miRNA biochip. The mimic was transfected into 4 highly metastatic breast cancer cell lines (BT549, HS578T, MDA-MB-231 and SUM159PT). The genes that were differentially expressed in highly metastatic breast cancer cell lines transfected with miR-200c mimic were detected by Aymetrix? MRNA biochip. Screening of miR-200c-regulated signal pathways and gene networks using the Capital Bio Molecule Annotation System (MAS). RESULTS: Nine differentially expressed miRNAs were screened from 12 breast cancer cell lines (P <0.01, multiple values ≥20 or ≤-20), of which miR-200c was most significantly down-regulated in highly metastatic breast cancer cell lines. A total of 33 co-up-regulated genes and 13 co-down-regulated genes were found in 4 miR-200c mimic-transfected breast cancer cell lines. The results of real-time fluorescence quantitative PCR and Western blotting showed that the mRNA and protein of zinc finger E-box binding homeobox 1 (ZEB1) were down-regulated in the four transfected cell lines. MAS bioinformatics analysis showed that the differential signaling pathways common in breast cancer cell lines transfected with miR-200c mimic include the olfactory transduction pathway, the cytokine-cytokine receptor (cytokine-cytokine receptor) interaction pathways and cell adhesion molecules (CAMs) pathways. Different signaling pathways can be linked through co-transcriptional genes, and there are close inter-related genes in specific signaling pathways. Conclusion: Based on the high-throughput biochip assay, bioinformatics analysis and multiplex screening of miR-200c gene networks provide a clear direction for further research on the mechanism of miR-200c.