先天性脊柱侧凸患者骨髓间充质干细胞差异表达microRNAs的鉴定分析

Identification of differentially expressed microRNAs in bone marrow mesenchymal stem cells from congenital scoliosis patients

目的:分析先天性脊柱侧凸(CS)患者和健康人骨髓间充质干细胞中的差异microRNAs(miRNAs)表达谱。方法:获取CS患者(椎体形成障碍亚组、椎体分节障碍亚组)及健康对照组的骨髓,分离培养、鉴定骨髓间充质干细胞,提取总RNA后标记miRNAs,使用微阵列芯片技术检测三组受试者骨髓间充质干细胞中的miRNAs表达谱,并对差异miRNAs进行生物信息学分析,通过qRT-PCR技术对获得的差异miRNAs进行验证。结果:在椎体形成障碍亚组和健康对照组之间鉴定出29个差异表达miRNAs,在椎体分节障碍亚组和健康对照组之间鉴定出27个差异表达miRNAs。两组差异表达miRNAs的交集共有10个。生物信息学分析结果显示,细胞内信号转导、高尔基体部分和蛋白质丝氨酸/苏氨酸激酶激活物活性分别是生物过程、细胞定位和分子功能中最显著的富集项,黏蛋白型O-聚糖的生物合成是所涉及的最重要的信号通路。qRT-PCR成功验证了其中2个差异表达的miRNAs:miR-412-3p和miR-766-5p。结论:本研究成功构建了CS患者与健康人骨髓间充质干细胞中的差异miRNAs表达谱。这些差异表达的miRNAs可能对阐述CS的发病机制具有重要意义,并有助于建立早期诊断生物标志物。

Objective: To analyze the microRNAs(miRNAs) expression profiles in bone marrow mesenchymal stem cells(MSCs) from congenital scoliosis(CS) patients and healthy controls. Methods: Bone marrow were obtained from CS patients(failure of formation, failure of segmentation) and healthy controls. After cell culture and identification of MSCs, total RNAs extraction and miRNAs labeling were performed. The expression profiles of miRNAs in three groups were detected by microarray technique.Bioinformatic analysis and qRT-PCR validation of these differentially expressed miRNAs were performed. Results: There were 29 differential miRNAs identified between the failure of formation group and healthy controls, and 27 differential miRNAs between the failure of segmentation group and healthy controls. Ten miRNAs were generated as overlapping of the two sets. Gene Ontology enrichment indicated that intracellular signal transduction, Golgi apparatus and protein serine/threonine kinase activator activity were the most significantly enriched terms in biological process, cellular location, and molecular function. KEGG database yielded mucin type O-glycan biosynthesis was the most important pathway.Two differential mi RNAs, miR-412-3p and miR-766-5p, were validated by qRT-PCR. Conclusions: We analyzed the differential mi RNAs expression profiles in MSCs from CS patients and healthy controls. These findings may help to illustrate the pathogenesis of CS and aid in the establishment of early diagnostic biomarkers.