MicroRNA-214-5p/TGF-β/Smad2 signaling alters adipogenic differentiation of bone marrow stem cells in postmenopausal osteoporosis

Abstract

Postmenopausal osteoporosis (OPM) is a common type of osteoporosis in females. It is a systemic, chronic bone disease that presents as microstructure degradation of osseous tissue, decreased bone mineral density and increased osteopsathyrosis caused by hypoovarianism and reduced estrogen levels in the body following menopause. In the present study, the role of microRNA (miR)‑214‑5p in the regulation of the expression of bone marrow stem cells (BMSCs) was investigated, and its molecular mechanism of osteogenic induction in vitro was assessed. When dexamethasone‑induced adipogenic differentiation was performed, miR‑214‑5p expression was increased compared with the control group, as determined by RT‑qPCR. Furthermore, oil red O staining, RT‑qPCR and western blot analysis demonstrated that overexpression of miR‑214‑5p promoted adipogenic differentiation, inhibited alkaline phosphatase (ALP), runt‑related transcription factor 2 (Runx2), osteocalcin (OC) and collagen α‑1 (I) chain (COL1A1) mRNA expression, and suppressed transforming growth factor (TGF)‑β, phosphorylated (p)‑Smad2 and collagen type IV α1 chain (COL4A1) protein expression in BMSCs. Additionally, downregulation of miR‑214‑5p increased the ALP, Runx2, OC and COL1 mRNA expression and increased TGF‑β, Smad2 and COL4A1 protein expression in BMSCs. Furthermore, a TGF‑β inhibitor was employed to inhibit TGF‑β expression in BMSCs following miR‑214‑5p downregulation, which led to reduced Smad2, TGF‑β and COL4A1 protein expression, and ALP, Runx2, OC and COL1 mRNA expression was also reduced, compared with the miR‑214‑5p downregulation only group. It was demonstrated that miR‑214‑5p may weaken osteogenic differentiation of BMSCs through regulating COL4A1. In conclusion, the results of the present study indicated that miR‑214‑5p may promote the adipogenic differentiation of BMSCs through regulation of the TGF‑β/Smad2/COL4A1 signaling pathway, and potentially may be used to develop a novel drug for postmenopausal osteoporosis.

Keywords: microRNA-214-5p; adipogenic differentiation; bone marrow stem cells; transforming growth factor-β; Smad2.

Figure 1.

Expression of miR-214-5p in dexamethasone-induced adipogenic differentiation of human bone marrow stem cells. (A) mRNA expression of miR-214-5p was determined by reverse transcription-quantitative polymerase chain reaction. (B) Successful adipogenic differentiation was determined using oil red O staining following dexamethasone treatment. Magnification, ×100. ##P<0.01 vs. control group. miR, microRNA.

Figure 2.

Adipogenic differentiation was measured by oil red O staining following miR-214-5p downregulation/overexpression in dexamethasone-treated cells. (A) Overexpression of miR-214-5p induced adipogenic differentiation in HBMSCs. (B) miR-214-5p downregulation inhibited adipogenic differentiation in HBMSCs. Magnification, ×100. miR, microRNA; HBMSCs, human bone marrow stem cells; negative, negative control transfection group; miR-214-5p group, overexpression of miR-214-5p group; anti-miR-214-5p group, miR-214-5p downregulation group.

Figure 3.

Overexpression of miR-214-5p affected ALP, Runx2, OC and COL1A1 mRNA expression. (A) RT-qPCR was initially performed to confirm successful overexpression of miR-214-5p following transfection. RT-qPCR was subsequently performed to determine the effect of miR-214-5p overexpression on (B) ALP, (C) Runx2, (D) OC and (E) COL1 mRNA expression in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OC, osteocalcin; COL1A1, collagen type IV α1 chain; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; negative, negative control transfection group; miR-214-5p group, overexpression of miR-214-5p group.

Figure 4.

Downregulation of miR-214-5p affected ALP, Runx2, OC and COL1A1 mRNA expression. (A) RT-qPCR was initially performed to confirm successful downregulation of miR-214-5p following transfection. RT-qPCR was subsequently performed to determine the effect of anti-miR-214-5p on (B) ALP, (C) Runx2, (D) OC and (E) COL1 mRNA expression in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OC, osteocalcin; COL1A1, collagen type IV α1 chain; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group.

Figure 5.

Overexpression of miR-214-5p affected the mRNA expression of PPARγ, CEBPα and adiponectin. Reverse transcription-quantitative polymerase chain reaction demonstrated that miR-214-5p overexpression increased the mRNA levels of (A) PPARγ, (B) CEBPα and (C) adiponectin in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; PPARγ, peroxisome proliferator-activated receptor γ; CEBPα, CCAAT/enhancer-binding protein α; negative, negative control transfection group; miR-214-5p group, overexpression of miR-214-5p group.

Figure 6.

Downregulation of miR-214-5p affected the mRNA expression of PPARγ, CEBPα and adiponectin. Reverse transcription-quantitative polymerase chain reaction demonstrated that miR-214-5p downregulation reduced the levels of (A) PPARγ, (B) CEBPα and (C) adiponectin in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; PPARγ, peroxisome proliferator-activated receptor γ; CEBPα, CCAAT/enhancer-binding protein α; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group.

Figure 7.

Overexpression of miR-214-5p suppressed COL4A1, TGF-β and p-Smad2 protein levels. (A) Western blot analysis demonstrated that miR-214-5p overexpression reduced the protein levels of COL4A1, TGF-β and p-Smad2. Densitometric analysis of western blotting results was performed to quantify the protein expression of (B) COL4A1, (C) TGF-β and (D) p-Smad2 following overexpression of miR-214-5p in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; COL4A1, collagen type IV α1 chain; TGF-β, transforming growth factor-β; p-Smad2, phosphorylated-Smad2; negative, negative control transfection group; miR-214-5p group, overexpression of miR-214-5p group.

Figure 8.

Downregulation of miR-214-5p enhanced the protein expression of COL4A1, TGF-β and p-Smad2. (A) Western blot analysis demonstrated that miR-214-5p downregulation increased the protein levels of COL4A1, TGF-β and p-Smad2. Densitometric analysis of western blotting results was performed to quantify the protein expression of (B) COL4A1, (C) TGF-β and (D) p-Smad2 following downregulation of miR-214-5p in human bone marrow stem cells. ##P<0.01 vs. negative control group. miR, microRNA; COL4A1, collagen type IV α1 chain; TGF-β, transforming growth factor-β; p-Smad2, phosphorylated-Smad2; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group.

Figure 9.

TGF-β inhibitor reversed the effects of miR-214-5p downregulation on the protein expression of COL4A1, TGF-β and p-Smad2. (A) Western blot analysis demonstrated that TGF-β inhibitor reversed anti-miR-214-5p-induced increases in the protein expression of COL4A1, TGF-β and p-Smad2. Densitometric analysis of western blotting results was performed to quantify the protein expression of (B) COL4A1, (C) TGF-β and (D) p-Smad2 following downregulation of miR-214-5p with or without TGF-β inhibitor treatment in human bone marrow stem cells. (E) Following miR-214-5p downregulation with or without TGF-β inhibitor treatment, adipogenic differentiation was assessed using oil red O staining. Magnification, ×100. ##P<0.01 vs. negative control group; **P<0.01 vs. anti-miR-214-5p group. TGF-β, transforming growth factor-β; miR, microRNA; COL4A1, collagen type IV α1 chain; p-Smad2, phosphorylated-Smad2; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group; TGF-β inhibitor group, miR-214-5p downregulation+TGF-β inhibitor group.

Figure 10.

TGF-β inhibitor reversed the effects of miR-214-5p downregulation on the mRNA expression of ALP, Runx2, OC and COL1. Reverse transcription-quantitative polymerase chain reaction demonstrated that TGF-β inhibitor reversed the anti-miR-214-5p-induced increases in the mRNA expression of (A) ALP, (B) Runx2, (C) OC and (D) COL1 in human bone marrow stem cells. ##P<0.01 vs. negative control group; **P<0.01 vs. anti-miR-214-5p group. TGF-β, transforming growth factor-β; miR, microRNA; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OC, osteocalcin; COL1A1, collagen type IV α1 chain; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group; TGF-β inhibitor group, miR-214-5p downregulation+TGF-β inhibitor group.

Figure 11.

TGF-β inhibitor reversed the effects of miR-214-5p downregulation on the mRNA expression of PPARγ, CEBPα and adiponectin. Reverse transcription-quantitative polymerase chain reaction demonstrated that TGF-β inhibitor reversed the anti-miR-214-5p-induced reduction in the mRNA expression of (A) PPARγ, (B) CEBPα and (C) adiponectin in human bone marrow stem cells. ##P<0.01 vs. negative control group; **P<0.01 vs. anti-miR-214-5p group. TGF-β, transforming growth factor-β; miR, microRNA; PPARγ, peroxisome proliferator-activated receptor γ; CEBPα, CCAAT/enhancer-binding protein α; negative, negative control transfection group; anti-miR-214-5p group, miR-214-5p downregulation group; TGF-β inhibitor group, miR-214-5p downregulation + TGF-β inhibitor group.