摘要
目的筛查miR-21抑制表达的人神经母细胞瘤细胞株（SK-N-SH1381）mRNA表达谱，寻找miR-21潜在下游靶基因，以进一步深入探讨miR-21及其参与的通路在肿瘤发生发展中的作用。方法采用Agilent Human Gene Expression Chip技术筛选了慢病毒转染稳定抑制miR-21表达的人神经母细胞瘤细胞株（SK-N-SH1381）和其配对的慢病毒转染无义序列的人神经母细胞瘤细胞株（SK-N-SHlv3-nc），运用生物信息技术，预测miR-21靶基因、基因功能富集，结合相关文献检索，筛选出候选基因，并在细胞水平通过荧光定量PCR验证芯片结果。结果基因表达谱芯片共筛选出4 985个差异在2倍以上的mRNA（P〈0.05），其中上调表达的基因2 734个，下调表达的基因2 251个。基于microRNA的作用，我们仅选择其中上调的2 734个基因为miR-21可能靶基因，与生物信息学软件分析预测出的398个潜在靶基因进行Venn分析，共获取64个候选潜在靶基因。进一步通过生物信息学分析与文献检索，挑选出6个可能靶基因，分别为EVA1A、TOM1L2、RGMA、LINC-PINT、NACC2及RPAP3。细胞水平荧光定量PCR结果均与芯片结果一致，EVA1A、TOM1L2是其中差异表达倍数变化最大的2个候选基因。结论结合芯片技术和生物信息学分析，在miR-21抑制表达的神经母细胞瘤细胞株中，筛选出6个miR-21可能的下游靶基因，分别为EVA1A、TOM1L2、RGMA、NACC2、RPAP3及LINC-PINT。其中EVA1A、TOM1L2是其中差异表达倍数变化最大的2个候选基因，进一步后续研究，探讨其在神经母细胞瘤发生发展中的作用。
Objective To explore mRNA expression profiling in a stable rniR-21 knockdown human neuroblastoma cell line （SK-N-SH13sl ） for screening potential downstream target genes of miR-21. Methods Human neuroblastoma cell line （SK-N-SH^138l） with stable miR-21 knockdown via lentiviral transfection was screened for changes in mRNA levels using Agilent Human Gene Expression Chips in comparison with the same cell line transfected with nonsense sequence （SK-N-SH^lv3-nc）. At the same time, we conducted in silico miR-21 targeting gene prediction, enrichment function of genes screened by chips and literature review to aid the selection of candidate genes. Then fluorescent quantitative polymerase chain reaction （PCR） was performed for verifying the results of chips at cellular level. Results Using mRNA chips, we identified 4985 mRNAs with a change of 〉2 folds （P 〈0. 05）. There were 2734 up-regulated and 2251 down-regulated genes. Based on the function of miRNAs, only the up-regulated genes were selected. After Venny analysis combining the results of chip and 398 potential target genes as predicted in silico, 64 candidate genes were identified as potential targets of miR-21. After further bioinformatic analysis and literature review, our selection was limited to 6 target genes of EVA1A, TOMIL2, RGMA, LINC-PINT, NACC2 and RPAP3. Quantitative PCR showed that the expressions of these genes were consistent with the findings of mRNA chips. Among 6 genes, EVA1A and TOMIL2 had the highest fold changes of differential expression. Conclusions With a combination of gene chip and bioinformatic analysis, 6 target genes of miR-21, i. e. EVA1A, TOM1L2, RGMA, NACC2, RPAP3 and LINC-PINT, are selected from human neuroblastoma ceil line with stable miR-21 knockdown. And EVA1A and TOM1L2 show the highest {old changes of differential expression. Further studies are required for exploring their roles during tumorigenesis.
出处
《中华小儿外科杂志》
CSCD
2016年第11期867-871,共5页
Chinese Journal of Pediatric Surgery
关键词
神经母细胞瘤
基因表达芯片
基因表达谱
Neuroblastoma
Gene expression chips
Gene expression profiling
