摘要
目的检测组蛋白去乙酰化酶7（HDAC7）是否为MicroRNA（miRNA）-140的靶基因，并研究在大鼠骨髓间充质干细胞（ratMSCs）成软骨诱导分化中miRNA-140和HDAC7表达的相关性。方法利用生物信息学、双荧光素酶报告基因检测以及Western blot法鉴定miRNA-140与HDAC7之间的靶作用关系。选取4周龄Sprague—Dawley（SD）雄性大鼠采用全骨髓贴壁法进行体外分离培养纯化得到大鼠骨髓间充质干细胞并向成软骨方向诱导分化，同时采用实时定量PCR和Western blot法检测miRNA-140和HDAC7的表达水平。结果双荧光素酶活性分析结果提示，miRNA-140靶结合人HDAC7基因的3’UTR。将miRNA-140mimics转染大鼠骨髓间充质干细胞后抑制HDAC7的蛋白表达。在大鼠骨髓间充质干细胞成软骨诱导分化过程中，miRNA-140表达上调，而HDAC7表达下降。结论miRNA-140通过靶结合HDAC7基因的3’UTR抑制HDAC7表达，二者在大鼠骨髓间充质干细胞成软骨诱导分化过程中表达负相关，提示miRNA-140通过抑制HDAC7蛋白表达保护软骨组织。
Objective To validate that histone deacetylases 7 （HDAC7） is a target of MicroRNA （miRNA） 140 and to investigate the correlation between miRNA-140 and HDAC7 expression in ehondrogenesis of rat bone marrow stromal cells （rat MSCs）. Methods We verified HDAC7 as a miRNA-140 target using bioinformatics, dual lueiferase reporter assay and Western blot analysis. MSCs from 4-week-old male Sprague-Dawley（SD） rats were isolated ,cultured and purified by the whole bone marrow adherence method. The expres- ion of miRNA-140 and HDAC7 were detected by using real-time quantitative PCR and Western blot analysis. Results Dual luciferase reporter assay showed that miRNA-140 could target human HDAC7 3＇UTR. The trans- fection of miRNA-140 mimics into rat MSCs interfered with the translation of HDAC7. The expression of miR-NA-140 was increased during chondrogenesis,and HDAC7 was downregulated. Conclusion miRNA-140 inter-acts with HDAC7 3＇ UTR to repress post-transcriptional HDAC7 expression,and the expression of miRNA-140 and HDAC7 are negatively correlated in chondrogenesis of rat MSCs. Our study reveals inhibiting HDAC7 ex-pression by miRNA-140 may contribute to the protection of cartilage tissue.
出处
《国际免疫学杂志》
CAS
2013年第6期462-466,共5页
International Journal of Immunology
基金
国家自然科学基金（81071480）
