长链非编码RNA AK089560在间质干细胞成骨与成脂分化中的表达  

The expression of long non-coding RNA AK089560 in mesenchymal stem cells undergoing osteogenic and adipogenic differentiation

作　　者：左长清[1] 卢旱云[1] 钟月春[1] 汪宗桂[2] 戴忠[1] 刘钰瑜[1] 吴铁[1] 

机构地区：[1]广东医学院药理学教研室,广东省东莞市523808 [2]广东医学院生物化学教研室,广东省东莞市523808

出　　处：《中国组织工程研究》2014年第23期3732-3738,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81101357;81102450);东莞市高等院校科研机构科技项目(2011108102029)~~

摘　　要：背景:最近研究发现众多长链非编码RNA调控干细胞多潜能性和分化。长链非编码RNA AK089560在干细胞多向分化中的表达变化及作用,目前尚不清楚。目的:观察间质干细胞C3H10T1/2成骨分化与成脂分化过程中AK089560的表达。方法:重组人骨形态发生蛋白2诱导间质干细胞C3H10T1/2成骨分化,碱性磷酸酶染色鉴定早期成骨分化。地塞米松、吲哚美辛和胰岛素三因子联合诱导C3H10T1/2成脂分化,油红O染色鉴定成脂分化结果。采用qRT-PCR检测诱导前后不同时间点AK089560表达的变化。采用RNAfold软件预测AK089560二级结构,UCSC基因组浏览器分析AK089560邻近编码蛋白基因,fancyGENE在线软件构件长链非编码RNA与编码基因关系图。结果与结论:C3H10T1/2经成骨诱导后,70%以上细胞碱性磷酸酶染色阳性;成脂分化诱导后,80%以上细胞油红O染色阳性。qRT-PCR结果显示,长链非编码RNA AK089560在成骨分化与成脂分化第2,4,6天表达均明显下降,成骨分化与成脂分化第2,4,6天与相应时间未分化组相比均差异有显著性意义(P<0.05)。生物信息学分析表明,LncRNA AK089560具有复杂茎环结构,与编码基因Sema3a形成sense overlap关系。结果表明AK089560在间质干细胞成骨与成脂分化中下调表达,提示其可能参与调控间质干细胞多向分化。BACKGROUND：Recent studies have found that stem cellpluripotency and differentiation is regulated by many long non-coding RNAs （LncRNAs）. The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear. OBJECTIVE：To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation. METHODS：Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors （dexamethasone, indomethacin and insulin） and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software. RESULTS AND CONCLUSION：Over 70%of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80%of the cells positive for oil red O staining after adipogenic induction. qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group （P〈0.05）. Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells.

关 键 词：干细胞 分化 间质干细胞 成骨分化 成脂分化 长链非编码RNA 生物信息学 重组人骨形态发生蛋白2 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]