Abstract:

Numerous studies indicated that microRNAs are critical in the regulation of cellular differentiation, by controlling the expression of underlying genes. The aim of this study was to investigate the effect of miR-210 upregulation on differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into osteoblasts. MSCs were isolated from HUCB and confirmed by their adipogenic/osteogenic differentiation and flow cytometric analysis of surface markers. Pre-miR-210 was amplified from human DNA, digested and ligated with plenti-III-mir-green fluorescent protein (GFP) vector, and cloned in STBL4 bacteria. After confirmation with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the plenti-III-GFP segment bearing pre-miR-210 was transfected into MSCs by electroporation. Two control vectors, pmaxGFP and Scramble, were transfected separately into MSCs. The expression of miR-210 and genes related to osteoblast differentiation, i.e., runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin gene, in the three groups of transfected MSCs was analyzed 0, 7, 14, and 21 days of transfection by quantitative reverse transcription PCR (qRT-PCR). Overexpression of miR-210 was observed in MSCs transfected with miR-210-bearing plasmid, and this was significantly different compared to Scramble group (p < 0.05). Significantly increased expression of Runx2 (at day 7 and 14), ALP and osteocalcin genes (at all time points for both genes) was observed in MSCs with miR-210-bearing plasmid compared to controls. Overall, the overexpression of miR-210 in MSCs led to MSC differentiation into osteoblasts, most probably by upregulating the Runx2, ALP, and osteocalcin genes at different stages of cell differentiation. Our study confirms the potential of miRNAs in developing novel therapeutic strategies that could target regulatory mechanisms of cellular differentiation in various disease states.

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