胰腺癌干细胞差异microRNAs的表达及生物信息学

Identification of differentially expressed microRNAs in pancreatic cancer stem cells

目的:筛选与胰腺癌干细胞相关的差异表达miRNAs,并进行生物信息学分析.方法:以MIA-PaCa2(TIChigh)与BxPc-3(TIClow)为研究胰腺癌干细胞的工具细胞制备总RNA,经质量鉴定后进行荧光标记;采用Agilent人类miRNA芯片进行杂交实验,获得miRNA表达谱;以Gene Spring Software 11.0软件和Quantile算法分析芯片实验数据,筛选与胰腺癌干细胞相关的差异miRNAs;荧光定量PCR验证部分差异表达miRNAs;基于Sanger数据库预测差异miRNA靶基因,基于Gene Ontology数据库进行GO注释,基于KEGG数据库进行Pathway注释.结果:获得符合基因芯片实验质量标准的RNA样品,基因芯片杂交及数据分析共鉴定到940个miRNAs,得到91个差异表达miRNAs(P<0.05,fold change≥2),其中MIAPaCa2(TIChigh)中下调表达45个,上调表达46个.荧光定量PCR验证21条差异miRNAs,其中19条均与芯片结果相符.TargetScan6.0数据库靶基因预测共得到2895条靶基因,GO注释显示他们主要涉及轴突导向、血管生成、转录后蛋白修饰等功能.Pathway注释显示主要涉及癌症途径、细胞-基质黏附途径、Hedgehog信号途径、MAPK信号途径等信号通路.结论:筛选出的胰腺癌干细胞相关差异表达miRNAs调控多种具有不同细胞功能和参与不同信号通路的基因,有可能成为胰腺癌新的治疗靶点.

AIM： To screen and identify differentially ex- pressed miRNAs in pancreatic cancer stem cells and to analyze them by bioinformatics.METHODS： Total RNA was prepared from MIA-PaCa2 （TIC^high） and BxPc-3 （TIC^high） cells. After quality identification and fluorescent la- beling, the RNA samples were hybridized with Agilent human miRNA microarrays. Raw data were normalized using Quantile algorithm and Gene Spring Software 11.0. Part of differentially expressed miRNAs were validated by SYBR Green real-time PCR. Sanger database was used to predict target genes for differentially expressed miRNAs. Gene Ontology database and KEGG database were used for GO annotations and Path- way annotations of target genes, respectively. RESULTS： A total of 940 miRNAs were iden- tified and among them 91 were differentially expressed （P 〈 0.05, fold change≥2）, includ- ing 45 up-regulated and 46 down-regulated ones in MIA-PaCa2 （TIC^high）. Twenty-one miR- NAs were validated by real-time PCR, and the results for 19 miRNAs were consistent with those of chip detection. 2 895 target genes were predicted for 91 differentially expressed miR- NAs, and these genes were mainly involved in events, such as axon guidance, angiogenesis, and post-translational protein modification, and in pathways such as cancer, focal adhesion, Hedgehog signaling pathway, and MAPK sig- naling pathway. CONCLUSION： Many differentially expressed miRNAs in pancreatic cancer stem cells have been identified, and their target genes are in- volved in a variety of biological events and in multiple signal pathways. These differentially expressed miRNAs may become new therapeu- tic targets for pancreatic cancer.