人根尖牙乳头干细胞与非干细胞差异表达miRNAs谱及靶基因的功能分析

Differential expression of target genes and gene function of miRNAs in human stem cells and non-stem cells from apical papilla

目的:比较人根尖牙乳头干细胞(SCAP)与非干细胞(非SCAP)之间差异表达的miRNAs,并进行功能预测分析。方法:采用酶消化法获得原代人根尖牙乳头细胞,用鼠抗人STRO-1单克隆抗体对其标记,用免疫磁珠分选系统获得SCAP和非SCAP。茜素红、油红O染色鉴定干细胞的分化能力;采用二代测序技术,检测SCAP和非SCAP中miRNAs的表达谱,并筛选差异表达(相差倍数>1.5)的miRNAs,用生物信息学方法对差异表达的miRNAs进行靶基因功能分析。结果:与非SCAP相比,SCAP中表达下调的miRNAs有hsamiR-4532、hsa-miR-6131、hsa-miR-4284、hsa-miR-3182、hsa-miR-1273e、hsa-miR-7-1-3p、hsamiR-31-3p、hsa-miR-3654、hsa-miR-495-3p、hsa-miR-382-3p,表达上调的miRNAs有hsa-miR-4508、hsa-miR-1247-5p、hsa-miR-6819-3p、hsa-miR-2116-3p、hsa-miR-490-5p、hsa-miR-335-5p、hsa-miR-1914-5p、hsa-miR-1249、hsa-miR-3180-3p、hsa-miR-668-3p。表达下调miRNAs所调控的下游靶基因主要有20个,表达上调的有6个,这些靶基因主要参与了细胞的自我更新、分化和迁移等生命过程。结论:SCAP特异性表达的miRNAs主要调控的下游靶基因及其基因功能可能与干性维持密切相关。

AIM： To study the differential expression of target genes and gene function of miRNAs in human stem cells and non-stem cells from apical papilla （SCAPs and NSCPs）.METHODS： SCAPs were isolated by immune-magnetic binding specific STRO-1 antibody separation system.Osteogenic and adipogenic differentiation of SCAPs were tested by alizarin red staining（ARS） and Oil Red O staining.Differential miRNAs of SCAPs and NSCPs were screened by the Next generation sequencing.Target genes and their possible roles were predicted using biological information.RESULTS： The results revealed that 10 miRNAs （hsa-miR-4532, hsa-miR-6131, hsa-miR-4284, hsa-miR-3182, hsa-miR-1273e, hsa-miR-7-1-3p, hsa-miR-31-3p, hsa-miR-3654, hsa-miR-495-3p, hsa-miR-382-3p） were downregulated while 10 miRNAs （hsa-miR-4508, hsa-miR-1247-5p, hsa-miR-6819-3p, hsa-miR-2116-3p, hsa-miR-490-5p, hsa-miR-335-5p, hsa-miR-1914-5p, hsa-miR-1249, hsa-miR-3180-3p, hsa-miR-668-3p） were upregulated in SCAPs compared with those of NSCPs （P〈0.05）.26 target genes which mainly involved in the self-renewal capability, cell differentiation and migration were found.CONCLUSION： The target genes and gene function regulated to specific miRNAs of SCAPs are closely related to the cell stemness.