miRNA-720 controls stem cell phenotype, proliferation and differentiation of dental pulp cells

Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs. Two cell types (dental pulp cells(DPCs), periodontal ligament cells(PDLCs)) were sorted by FACS to isolate side population (SP) and main population (MP) cells. Then a microRNA array was performed with the SP and MP cells of DPCs and PDLCs.

牙髓细胞（Dental pulp cells, DPCs）以富含干/祖细胞而为人熟知，但其特征仍未被充分阐明。小型非编码微小RNA（small non-coding microRNAs, miRNAs）已被证实可调控蛋白质翻译、mRNA稳定性及转录过程，且有研究表明其在干细胞生物学中发挥重要作用，与细胞重编程、干性维持及细胞分化调控密切相关。为阐明牙髓干/祖细胞的特征及其分化机制，本研究从人类牙髓细胞及牙周膜细胞（periodontal ligament cells, PDLCs）中分选得到富集干细胞的侧群（side population, SP）细胞，并开展了基于锁核酸（locked nucleic acid, LNA）的miRNA芯片检测。结果显示，与侧群细胞相比，miR-720在分化型主群（main population, MP）细胞中呈高表达。计算机模拟分析（in silico analysis）与报告基因实验（reporter assay）证实，miR-720可靶向干细胞标志物NANOG，提示miR-720可能通过抑制NANOG的表达促进牙髓干/祖细胞的分化。确实，功能获得与功能缺失实验（gain-and loss-of-function analyses）表明，miR-720可调控NANOG的转录本及蛋白水平。此外，转染（transfection）miR-720可显著降低早期干细胞标志物SSEA-4阳性细胞的数量。与此同时，已知在干细胞分化过程中发挥关键作用的DNA甲基转移酶（DNA methyltransferases, DNMTs）的mRNA水平，也可通过miR-720上调，但其具体机制尚未明确。最终，通过针对Ki-67的免疫细胞化学分析（immunocytochemical analysis）证实，miR-720可抑制牙髓细胞的增殖；同时茜素红染色（alizarin red staining）、碱性磷酸酶（alkaline phosphatase）及骨桥蛋白（osteopontin）的mRNA水平检测结果表明，miR-720可促进成牙本质分化（odontogenic differentiation）。本研究证实miR-720是一种调控牙髓细胞分化的新型微小RNA。本研究通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）对两种细胞类型（牙髓细胞DPCs、牙周膜细胞PDLCs）进行分选，以分离侧群细胞与主群细胞。随后针对牙髓细胞与牙周膜细胞的侧群、主群细胞开展了miRNA芯片检测。