The potential effect of BMSCs with miR-27a in improving steroid-induced osteonecrosis of the femoral head

Abstract

Steroid induced osteonecrosis of the femoral head (ONFH) frequently leads to femoral head collapse and subsequent hip arthritis. This study aimed to investigate the potential therapeutic mechanism of miR-27a on steroid-induced ONFH. Levels of IL-6, TNF-α, miR-27a, Runx2, PPAR-γ and ApoA5 were first examined in bone marrow tissues from steroid-induced ONFH and controls. Subsequently, we overexpressed or knocked down miR-27a in bone marrow mesenchymal stem cells (BMSCs) and detected cell proliferation, osteogenic differentiation, adipogenic differentiation. In addition, miR-27a mimics and BMSCs were injected into the established steroid-induced ONFH rats, and the osteoprotective effects of both were evaluated. Dual luciferase reporter was used to test the targeting effect of miR-27a-3p and PPARG. miR-27a and Runx2 were lowly expressed in steroid-induced ONFH, PPAR-γ and ApoA5 were highly expressed. Overexpression of miR-27a in BMSCs promoted cell proliferation and osteogenic differentiation, inhibited adipogenic differentiation. Furthermore, increasing miR-27a and BMSCs obviously reduced bone loss in steroid induced ONFH rats. The expressions of Runx2 in BMSCs and steroid-induced ONFH rats was significantly up-regulated, while IL-6, TNF-α, PPAR-γ and ApoA5 were down-regulated with miR-27a overexpression. Additionally, PPARG was the target of miR-27a-3p. The results of the present study reveal a role for miR-27a in promoting osteogenesis and may have a synergistic effect with BMSCs.

Figure 1

The levels of miR-27a, Runx2, PPAR-γ and ApoA5 in steroid-induced ONFH patients. (A) Comparison of miR-27a mRNA level between steroid-induced ONFH patients and controls. (B) Comparison of Runx2, PPAR-γ and ApoA5 mRNA level between steroid-induced ONFH patients and controls. (C) Protein expression of Runx2, PPAR-γ and ApoA5 between steroid-induced ONFH patients and controls. ***P < 0.001.

Figure 2

Overexpression of miR-27a promoted cell proliferation and changed expression of downstream genes. (A) Determination of optimal conditions for lentiviral transfection of BMSCs. (B) After transfection with miR-27a mimics, miR-27a inhibitor, and NC, the expression of miR-27a was detected by qRT‐PCR. (C) After transfection with miR-27a mimics, miR-27a inhibitor, and NC, the expression of Runx2, PPAR-γ and ApoA5 were detected by qRT‐PCR. (D) After transfection with miR-27a mimics, protein expression of Runx2, PPAR-γ and ApoA5 were detected by WB. **P < 0.01, ***P < 0.001. (E) The cell proliferation of BMSCs with transfected miR-27a mimics, miR-27a inhibitor, and NC were assessed by CCK-8 assay. (F) Luciferase activity decreased following co-transfection with miR-27a-3p mimic and wild-type (WT) PPARG-3′-UTR luciferase plasmid.

Figure 3

Osteogenic differentiation and adipogenic differentiation of BMSCs effected by miR-27a. (A) Alizarin red staining of BMSCs after osteogenic induction (OI). Scale bar, 50 μm. (B) The ALP activity of BMSCs after osteogenic induction (OI). **P < 0.01 vs BMSCs, ##P < 0.01 vs BMSCs after OI. (C) The oil red O staining of BMSCs after adipogenic induction (AI). Scale bar, 20 μm. (D) Number of oil red O-positive cells in different groups. **P < 0.01 vs BMSCs, ##P < 0.01 vs BMSCs after AI.

Figure 4

miR-27a protect bone tissue in steroid-induced ONFH rats. (A) Micro-CT scanning images of femoral head of rat models. (B) The BS/BV, BV/TV, Tb.N, Tb.Th, and Tb.Sp values of rat femoral heads in different groups. **P < 0.01 vs controls, ##P < 0.01 vs steroid-induced ONFH. (C) Hematoxylin–eosin staining of femoral head of rat models. Black arrows, dipocyte; red arrows, empty bone lacunae.

Figure 5

Overexpression of miR-27a changed gene expression associated with inflammation, adipogenic and osteogenic differentiation in steroid-induced ONFH rat. (A) The levels of IL-6 and TNF-α in serum of rats were detected using ELISA. (B)The mRNA levels of miR-27a, Runx2, PPAR-γ and ApoA5 were detected by qRT‐PCR in different group rats. (C) The protein expression of miR-27a, Runx2, PPAR-γ and ApoA5 were detected by WB in different group rats. *P < 0.05, **P < 0.01, ***P < 0.001.