人骨髓间充质干细胞成骨分化相关的长链非编码RNA表达谱特征

Expression profile of long non-coding RNAs involved in osteogenic differentiation of human marrow-derived mesenchymal stem cells

目的观察人骨髓间充质干细胞(h MSCs)成骨分化过程中长链非编码RNA(lncRNA)的表达谱变化。方法以h MSCs及成骨诱导分化7、14、21 d后的细胞为研究对象,利用Agilent lncRNA芯片技术筛选出成骨诱导分化前后2倍变化幅度的差异lncRNA,并通过实时荧光定量PCR(RT-PCR)对芯片结果进行验证。选取筛选结果中表达差异较大的lncRNA利用UCSC Genome Browser并结合芯片筛选结果分析lncRNA邻近蛋白编码基因。结果 h MSCs经成骨诱导分化7、14、21 d后,均具有成骨细胞特性;芯片筛选结果表明,与成骨诱导分化前的h MSCs相比,成骨诱导分化7、14、21 d后持续表达上调超过2倍的lncRNA有433条,下调超过2倍的有232条;选取其中部分lncRNA,RT-PCR验证与芯片结果相符合;对lncRNA邻近蛋白编码基因分析提示某些lncRNA(如lncRNA ENST00000585537.1和lncRNA e HIT00001595)可能在h MSCs成骨分化过程中发挥重要作用。结论 h MSCs成骨诱导分化前后,lncRNA表达谱发生显著变化,提示差异表达的lncRNA可能与h MSCs成骨分化密切相关,由此可进一步解释h MSCs成骨分化机制。

Objective To analyze the expression profile variation of long non- coding RNA（ lncRNA） during osteogenesis of human marrow- derived mesenchymal stem cells（ hMSCs）. Methods Osteoblast- induced differentiation was perfomed on hMSCs. Using Agilent lncRNA microarray,samples of hMSCs and differentiated hMSCs on the 7^th,14^th and 21^st day were assayed to identify the target lncRNA,which was altered more than 2 times during osteogenesis of hMSCs. The expression of lncRNA was verified by real time quantification- polymerase chain reaction（ RT- PCR）. Specific lncRNAs related protein- coding genes using the UCSC Genome Browser combined with microarray results. Results Osteogenic characteristics were observed in induced hMSCs on the 7^th,14^th and 21^st day. According to microarray analysis,433 lncRNAs were constantly significantly up- regulated more than 2 times and 232 lncRNAs constantly significantly down- regulated more than 2 times during the osteogenesis of hMSCs. The results of RT- PCR were consistent with miRNA microarray. Analysis of nearby protein- coding genes prompted that some lncRNAs,such as lncRNA ENST00000585537. 1and lncRNA e HIT00001595,may play important roles in osteogenesis of hMSCs. Conclusion During osteogenesis of hMSCs,lncRNA expression profiles have significantly changed,these different lncRNAs maybe closely related to the osteogenesis of hMSCs and these findings can also further explain the mechanism of osteogenesis of hMSCs.