MicroRNA-141 and miR-200a induce the chondrogenic cell fate in human periodontal ligament cells by targeting TWIST2 and KLF12

Abstract

Differentiation of mesenchymal stem cells (MSCs) in human periodontal ligaments (HPDLs) is induced by specific transcription factors that support periodontal tissue regeneration. The maintaining characteristics for HPDL cells could be regulated by two kinds of transcription factors, such as Twist family basic helix-loop-helix (bHLH) transcription factor 2 (TWIST2) and Krüppel-like factor 12 (KLF12). Because TWIST2 and KLF12 may be controlled by microRNAs (miRNAs), we investigated their 3′-untranslated regions (3′-UTRs) that contain seed sequences for miR-141 and miR-200a. HPDL cells were co-transfected with luciferase (LUC) reporter plasmids with or without 3′-UTRs for TWIST2 or KLF12, and miR-141 or miR-200a expression vectors. MiR-141 and miR-200a suppressed LUC activities via the TWIST2 or KLF12-3′-UTRs and reduced the mRNA and protein levels of TWIST2 and KLF12. These effects of miR-141 and miR-200a were related to reduced and increased expressions of the collagen Type I (COL1A1) and aggrecan (ACAN), while increasing the mRNAs for the chondrogenic factors SRY-Box transcription factor 5 (SOX5) SOX6, and Tricho-rhino-phalangeal syndrome type 1 (TRPS1) genes. We validated that these effects also reduce SOX5 and SOX6 proteins levels. Expression of miR-141 and miR-200a was significantly higher in HCS2/8 chondrogenic cells than human gingival fibroblasts (HGF), or HPDL and Saos2 cells. Furthermore, transfection of HPDL cells with miR-141 or miR-200a produced alcian blue staining. Thus, miR-141 and miR-200a directly target TWIST2 and KLF12 and induce chondrogenic differentiation of HPDL cells.