Physical Exercise Modulates miR-21-5p, miR-129-5p, miR-378-5p, and miR-188-5p Expression in Progenitor Cells Promoting Osteogenesis

Abstract

Physical exercise is known to promote beneficial effects on overall health, counteracting risks related to degenerative diseases. MicroRNAs (miRNAs), short non-coding RNAs affecting the expression of a cell's transcriptome, can be modulated by different stimuli. Yet, the molecular effects on osteogenic differentiation triggered by miRNAs upon physical exercise are not completely understood. In this study, we recruited 20 male amateur runners participating in a half marathon. Runners' sera, collected before (PRE RUN) and after (POST RUN) the run, were added to cultured human mesenchymal stromal cells. We then investigated their effects on the modulation of selected miRNAs and the consequential effects on osteogenic differentiation. Our results showed an increased expression of miRNAs promoting osteogenic differentiation (miR-21-5p, miR-129-5p, and miR-378-5p) and a reduced expression of miRNAs involved in the adipogenic differentiation of progenitor cells (miR-188-5p). In addition, we observed the downregulation of PTEN and SMAD7 expression along with increased AKT/pAKT and SMAD4 protein levels in MSCs treated with POST RUN sera. The consequent upregulation of RUNX2 expression was also proven, highlighting the molecular mechanisms by which miR-21-5p promotes osteogenic differentiation. In conclusion, our work proposes novel data, which demonstrate how miRNAs may regulate the osteogenic commitment of progenitor cells in response to physical exercise.

Keywords: PPARG2; RUNX2; miRNAs; physical exercise.

Figure 1

miR-21 expression was upregulated in MSCs treated with POST RUN sera during the differentiation (A); miR-129-5p was significantly upregulated only after 14 days of differentiation (B); MiR-188-5p was downregulated after 14 and 21 days of differentiation (C). In contrast, miR-378-5p resulted upregulated after 14 and 21 days of differentiation (D). Data were obtained from three independent experiments * p < 0.05 and ** p < 0.01.

Figure 2

Upregulation of RUNX2 was observed in MSCs treated with POST RUN compared to PRE RUN sera (A). Bone nodule formation ability assessed by Alizarin Red S was in POST RUN sera MCSs (B). Increased osteogenic differentiationin in POST RUN sera MSCs was confirmed by the upregulation of Collagen type I alpha 2 chain (COL1A2; C) and Integrin Binding Sialoprotein (IBSP D) as well as by increased protein levels of Osteocalcin (OCN) (E) Data were collected from three independent experiments. OD: optical density; * p < 0.05 and ** p < 0.01.

Figure 3

Cleaved caspase-3/caspase-3 ratio was reduced in MSCs treated with POST RUN sera (A) PPARG was downregulated in MSCs treated with POST RUN sera (B). The reduced adipogenic commitment was confirmed by a reduced number of lipid droplets for cells as well as the % of Oil Red O positive stained area (C). Data were collected from three independent experiments. * p < 0.05 and ** p < 0.01.

Figure 4

Downregulation of PTEN (A) and SMAD7 (B) mRNAs was observed in MSCs treated with POST RUN sera. Western Blot analyses showed increased levels of total AKT (C) as well as of SMAD4 proteins (D). Data were collected from three independent experiments. * p < 0.05 and ** p < 0.01.

Figure 5

A schematic view describing the role of physical exercise in promoting osteogenic differentiation by inducing miR-21-5p upregulation.