目的 通过研究Chir99021联合PD0325901对小鼠胚胎干细胞中miRNAs差异表达的影响,为揭示胚胎干细胞的自我更新和分化的机制提供更多线索.方法 采用miRNA基因芯片技术检测Chir99021联合PD0325901处理组和PD0325901处理组miRNAs的表达谱差异.选取3倍以上及通过查文献与胚胎干细胞自我更新相关的1.5倍以上3倍以下的miRNAs,采用实时荧光定量PCR法验证,利用miRDB、Miranda两个数据库交叉预测差异表达的靶基因,并应用KEGG Pathway进行靶基因功能富集分析.结果 与PD0325901单独处理相比,Chir99021联合PD0325901处理组有47种miRNAs上调1.5倍以上,75种miRNAs下调1.5倍以上;用实时荧光定量PCR验证差异表达的miRNAs,结果显示13个miRNAs与芯片结果相符.靶基因预测分析显示,miR-466a-5p、miR-466d-5p处于重要位置,Plcb1、Prkcb处于关键基因位置.结论 Chir99021可引起小鼠胚胎干细胞中的miRNAs差异表达,差异表达的miRNAs可能通过调控Plcb1、Prkcb基因而影响胚胎干细胞的自我更新.
Objectives To study the effect of Chir99021 combined with PD0325901 on differential expression of miRNAs in embryonic stem cells of mouse, and to provide more clues for revealing the mechanism of embryonic stem cell self-renewal and differentiation. Methods The miRNA chip method was used to detect the differences of miRNAs expression between Chir99021 combined with PD0325901 treatment group and PD0325901 treatment group. The miRNAs which differentiated from PD0325901 group more than 3 times and between 1.5 and 3 times but relating self-renewal through previous literature were selected. The differentially expressed miRNAs were then verified by qRT-PCR method. The target genes of differentially expressed miRNAs were cross-forecast by miRDB and Miranda databases, and functional rich products of these target genes were assayed by KEGG Pathway. Results Compared with PD0325901 treatment group, 47 miRNAs were up-regulated more than 1.5 times and 75 miRNAs were down-regulated more than 1.5 times in Chir99021 combined with PD0325901 treatment group. qRT-PCR method verified 13 miRNAs. MiroRNA-Gene-Network showed that 466d-5p and 466a-5p might be critical miRNAs and play a key role in Plcb1 and Prkcb gene expression. Conclusion Chir99021 can induce differentially expressed miRNAs in embryonic stem cells, which may affect the embryonic stem cells self-renewal through miRNAs-induced Plcb1 and Prkcb gene expression.