Expression profile of Parathyroid hormone-related protein (PTHrP) induced miRNAs during TGF-beta mediated chondrogenesis of marrow derived human mesenchymal stem cells

A miRNA microarray was used to identify candidate miRNAs regulated by PTHrP treatment in chondrogenic hMSC differentiation. miRNA-expression patterns after PTHrP treatment for 1 week (CM-T-Pt-L1) or 3 weeks (CM-T-Pt-3) in chondrogenic culture were compared with reference levels obtained from cell pellets treated with TGF-β only (CM-T). Under identical chondrogenesis conditions (except for PTHrP treatment), miRNA-expression patterns were relatively unchanged when compared with the TGF-β-only control, which simplified the selection of candidate miRNAs. Microarray analysis revealed that only 4 miRNAs in CM-T-Pt-L1 cells and 7 miRNAs in CM-T-Pt-3 cells were differentially expressed following PTHrP treatment. Hsa-miR-590-5p and hsa-miR-892b were commonly down-regulated or up-regulated in both PTHrP-treated groups. Interestingly, hsa-miR-892b expression was not detected in the TGF-β-only control, while hsa-miR-877*, hsa-miR-1288, and hsa-miR-1305 were up-regulated. Therefore, PTHrP treatment induced hsa-miR-892b expression during TGF-β-mediated hMSC chondrogenesis. hsa-miR-892b expression in CM-T-Pt-L1 cells was 2.43-fold higher than that in CM-T-Pt-3 cells. PTHrP induced miRNA expression in human MSC was measured at 21 days of chondrogenic induction after exposure for last 1 week or all 3 weeks to dose of 100 ng/ml PTHrP. Four independent experiments were performed depending on PTHrP-exposure times (0, 1, and 3 weeks) using two different donors for each experiment.

本研究采用miRNA微阵列（miRNA microarray），旨在筛选软骨分化的人间充质干细胞（human mesenchymal stem cell, hMSC）中受甲状旁腺激素相关蛋白（parathyroid hormone-related protein, PTHrP）调控的候选miRNA。将软骨培养体系中经PTHrP处理1周（CM-T-Pt-L1）或3周（CM-T-Pt-3）后的miRNA表达谱，与仅经转化生长因子-β（transforming growth factor-β, TGF-β）处理的细胞团块所获参照水平（CM-T）进行对比。在除PTHrP处理外其余条件完全一致的软骨分化体系中，miRNA表达谱与仅用TGF-β处理的对照组相比无显著变化，这大幅简化了候选miRNA的筛选流程。微阵列分析结果显示，经PTHrP处理后，CM-T-Pt-L1组细胞中仅4种miRNA存在差异表达，CM-T-Pt-3组细胞中则仅7种miRNA存在差异表达。hsa-miR-590-5p与hsa-miR-892b在两个PTHrP处理组中均呈现一致的上调或下调变化。值得注意的是，仅用TGF-β处理的对照组中未检测到hsa-miR-892b的表达，而hsa-miR-877*、hsa-miR-1288及hsa-miR-1305则呈现上调表达。因此，在TGF-β介导的hMSC软骨分化过程中，PTHrP处理可诱导hsa-miR-892b的表达。CM-T-Pt-L1组细胞中hsa-miR-892b的表达量较CM-T-Pt-3组高2.43倍。本研究还检测了在软骨诱导分化21天期间，于最后1周或全程3周以100 ng/ml剂量施加PTHrP处理后，人间充质干细胞中PTHrP诱导的miRNA表达水平。本研究共开展4次独立实验，实验分组依据PTHrP暴露时长（0、1及3周）设置，且每次实验均使用2名不同供者的细胞。