目的 通過研究Chir99021聯(lián)合PD0325901對小鼠胚胎干細(xì)胞中miRNAs差異表達(dá)的影響,為揭示胚胎干細(xì)胞的自我更新和分化的機(jī)制提供更多線索.方法 采用miRNA基因芯片技術(shù)檢測Chir99021聯(lián)合PD0325901處理組和PD0325901處理組miRNAs的表達(dá)譜差異.選取3倍以上及通過查文獻(xiàn)與胚胎干細(xì)胞自我更新相關(guān)的1.5倍以上3倍以下的miRNAs,采用實時熒光定量PCR法驗證,利用miRDB、Miranda兩個數(shù)據(jù)庫交叉預(yù)測差異表達(dá)的靶基因,并應(yīng)用KEGG Pathway進(jìn)行靶基因功能富集分析.結(jié)果 與PD0325901單獨處理相比,Chir99021聯(lián)合PD0325901處理組有47種miRNAs上調(diào)1.5倍以上,75種miRNAs下調(diào)1.5倍以上;用實時熒光定量PCR驗證差異表達(dá)的miRNAs,結(jié)果顯示13個miRNAs與芯片結(jié)果相符.靶基因預(yù)測分析顯示,miR-466a-5p、miR-466d-5p處于重要位置,Plcb1、Prkcb處于關(guān)鍵基因位置.結(jié)論 Chir99021可引起小鼠胚胎干細(xì)胞中的miRNAs差異表達(dá),差異表達(dá)的miRNAs可能通過調(diào)控Plcb1、Prkcb基因而影響胚胎干細(xì)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.