CircRNA_25487 inhibits bone repair in trauma-induced osteonecrosis of femoral head by sponging miR-134-3p through p21

Abstract

We aimed to identify specific circular RNAs (circRNAs) involved in bone repair of trauma-induced osteonecrosis of femoral head (TIONFH) and to explore the potential mechanism. CircRNA sequencing on the blood sample collected from patients with and without TIONFH was performed to select cirRNAs that were significantly differentially expressed, followed by qRT-PCR confirmation. Furthermore, the functions of one selected circRNA and the potential mechanisms in bone repair of TIONFH were validated based on the bone marrow mesenchymal stem cells (BMSCs) and osteoclast-like cells (OLCs) through CCK-8, flow cytometry, transwell assay, luciferase reporter assay, and western blot. A total of 234 upregulated and 148 downregulated differentially expressed circRNAs were identified, and qRT-PCR showed that circRNA_25487 was significantly upregulated in the peripheral blood of TIONFH patients. Luciferase reporter assay confirmed the binding effect between miR-134-3p and circRNA_25487. CircRNA_25487 suppression and miR-134-3p overexpression could promote cell proliferation and invasion while inhibited apoptosis of BMSCs and OLCs. miR-134-3p could target p21. CircRNA_25487 inhibited bone repair in TIONFH by sponging miR-134-3p to upregulate the expression of p21.

Keywords: Circular RNA; Proliferation; Trauma-induced osteonecrosis of femoral head; microRNA.

Fig. 1

MRI and X-ray examination of TIONFH patients and the patients without TIONFH. (A) X-ray of patients without necrosis showed a smooth surface without collapse. (B) MRI of patients without necrosis showed hollow spike-like images without a significant “double line”. (C) X-ray of necrosis patients showed femoral head collapse, density changes, cystic changes, or hollow or pierced femoral head surface. (D) MRI of necrosis patients showed a clear “double line” sign and displayed high signals on fat-suppressed images.

Fig. 2

(A) Heatmap of differentially expressed circRNAs. (B) Relative expression levels of circRNA_25775, circRNA_33798, circRNA_25487, circRNA_18052 and circRNA_3684 in blood samples of two groups detected by qRT-PCR.

Fig. 3

(A) The relative luciferase activities of six miRNAs, and (B) miR-134-3p had interaction with circRNA 25487-psicheck.

Fig. 4

(A and B) Relative expression levels of miR-134-3p in BMSCs and OLCs after cells were transfected with miR-134-3p mimic. (C and D) Proliferation rates of BMSCs and OLCs after circRNA_25487 suppression and miR-134-3p overexpression. (E and F) Invasion of BMSCs and OLCs after circRNA_25487 suppression and miR-134-3p overexpression. (G and H) Apoptosis rates of BMSCs and OLCs after circRNA_25487 suppression and miR-134-3p overexpression. ∗P < 0.05, ∗∗∗P < 0.001 compared with control.

Fig. 5

(A) The regulatory relation between miR-134-3p and p21. Co-transfection of miR-134-3p mimics and wild-type reporter gene plasmid significantly decreased the luciferase activity. (B and C) The mRNA and protein expression of p21 after circRNA_25487 suppression and miR-134-3p overexpression. ∗P < 0.05, ∗∗∗P < 0.001 compared with control.