Mesenchyme homeobox 1 mediated-promotion of osteoblastic differentiation is negatively regulated by mir-3064-5p

Abstract

Human mesenchymal stem cells (hMSCs) are multipotent cells that can be differentiated into different cell types including osteoblasts. Herein we aimed to assess the regulation of transcription factor mesenchyme homeobox 1 (Meox1) in the osteogenic differentiation of hMSCs and to determine the microRNA which targets on Meox1. Total RNA was extracted from the isolated ligamentum flavum tissue samples and cultured hMSCs, and the expression of Meox1 was assessed by RT-PCR and Western blot assays. Cultured hMSCs were induced towards osteoblastic differentiation, and the osteoblast phenotype was determined by alkaline phosphatase activity and alizarin red staining. The microRNA targeting on the 3′-UTR of Meox1was predicted using bioinformatics tool, and the binding was validated by luciferase and RNA pulldown assays. The osteoblastic differentiation of hMSCs was checked with the knockdown of Meox1 and microRNA inhibitors. Higher expression of Meox1, and lower expression of miR-3064–5p in ossified ligamentum flavum (OLF) tissues were identified. In addition, increased expression along with the osteoblastic differentiation of hMSCs was found. Further research revealed that Meox was a direct target of miR-3064–5p, when the former promoted the differentiation of hMSCs into osteoblasts, the latter significantly suppressed the osteogenesis. The expression of Meox1 increased gradually with the osteoblastic differentiation of hMSCs, during which miR-3064–5p decreased. Meox1 is a direct target of miR-3064–5p, and they both play important roles in the osteogenesis. These findings provide potential target for the development of therapeutic drugs for skeletal system diseases.