title: miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.
abstract: 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.
authors.affiliation: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Emilio Satoshi
authors.initials: ES
authors.lastname: Hara
authors.affiliation: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Ono
authors.affiliation: Department of Radiation Oncology, Division of Molecular and Cellular Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Takanori
authors.initials: T
authors.lastname: Eguchi
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubota
authors.affiliation: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Hai Thanh
authors.initials: HT
authors.lastname: Pham
authors.affiliation: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Sonoyama
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Tajima
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
authors.affiliation: Department of Radiation Oncology, Division of Molecular and Cellular Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Stuart K
authors.initials: SK
authors.lastname: Calderwood
authors.affiliation: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama-shi, Okayama-ken, Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Kuboki
mesh_terms: D020413:3' Untranslated Regions; D000328:Adult; D020029:Base Pairing; D001483:Base Sequence; D015415:Biomarkers; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D016000:Cluster Analysis; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D003782:Dental Pulp / Q000166:cytology* / Q000502:physiology*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D010641:Phenotype; D058985:Side-Population Cells / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D014158:Transcription, Genetic; D000097584:DNA Methyltransferase 3B
chemical_list: D020413:3' Untranslated Regions; D015415:Biomarkers; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D018398:Homeodomain Proteins; D035683:MicroRNAs; D000071317:Nanog Homeobox Protein; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C568636:MIRN720 microRNA, human; C486780:NANOG protein, human