SARS-CoV-2-induced Overexpression of miR-4485 Suppresses Osteogenic Differentiation and Impairs Fracture Healing

Abstract

The angiotensin-converting enzyme 2 (ACE2) receptor has been identified as the cell entry point for SARS-CoV-2. Although ACE2 receptors are present in the bone marrow, the effects of SARS-CoV-2 on the biological activity of bone tissue have not yet been elucidated. In the present study we sought to investigate the impact of SARS-CoV-2 on osteoblastic activity in the context of fracture healing. MicroRNA-4485 (miR-4485), which we found to be upregulated in COVID-19 patients, negatively regulates osteogenic differentiation. We demonstrate this effect both in vitro and in vivo. Moreover, we identified the toll-like receptor 4 (TLR-4) as the potential target gene of miR-4485, and showed that reduction of TLR-4 induced by miR-4485 suppresses osteoblastic differentiation in vitro. Taken together, our findings highlight that up-regulation of miR-4485 is responsible for the suppression of osteogenic differentiation in COVID-19 patients, and TLR-4 is the potential target through which miR-4485 acts, providing a promising target for pro-fracture-healing and anti-osteoporosis therapy in COVID-19 patients.

Keywords: Differentiation; Fracture; Osteoblast; SARS-CoV-2; miR-4485.

Figure 1

The differentially expressed miRNAs between IgG(+) and IgG(-) patients. (A) The cluster analysis diagram and (B) volcano diagram of the differentially expressed miRNAs.

Figure 2

The level of miRNAs between IgG(+) and IgG(-) patients. (A) The level of miR-4485-3p between IgG(+) and IgG(-) patients. (B) The level of miR-1973 between IgG(+) and IgG(-) patients. n=30, per group. Data are the mean ± SD 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

miR-4485-3p suppress osteogenic differentiation in vitro. (A) The level of miR-4485-3p was measured by qRT-PCR analysis. (B) The level of osteogenic-related mRNAs in different groups was measured by qRT-PCR analysis. (C) The level of osteogenic-related mRNAs in different groups was measured by western blot analysis. (D-E) The alizarin red staining and ALP staining results of different groups. Data are the mean ± SD 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

Toll-like receptor 4 (TLR4) is a potential target gene for miR-4485-3p. (A) The top 10 hub genes with the highest degree of interaction determined with Cytoscape (Centiscape 2.2 plugin) were CBL, CDC34, CRK, GMPS, PIK3C3, RAB7A, RNF41, STAT5B, TLR4, and UBE2K (Fig. 4A). (B-C) The top 10 genes associated with the enriched GO/KEGG pathway are illustrated using a chord diagram. (D) Expression of the top 50 genes from the 6 modules and their positions on chromosomes are also shown.

Figure 5

TLR4 is involved in miR-4485-3p-mediated osteogenic differentiation. (A) The luciferase assay of miR-4485-3p and TLR4. (B-E) The level of TLR-4 was measured by qRT-PCR and western blot analysis. (F-G) The level of osteogenic-related mRNAs in different groups was measured by western blot analysis. (H-I) The alizarin red staining and ALP staining results of different groups. Data are the mean ± SD 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

Overexpression of miR-4485-3p hinders fracture healing in vivo. (A) The animal micro-CT results of different groups. (B) The analysis of micro-CT parameters. (C) The level of TLR4 in the callus was assessed by qRT-PCR analysis, n=6, per group. *p<0.05, **p<0.01, ***p<0.001.

Figure 7

Local delivery of miR-4485-3p antagonist rescues the delayed fracture healing phenotype in mice. (A) Fractures in the siRNA-TLR4+AntagomiR-4485-3p-treated mice had reduced cartilage and increased bone compared with siRNA-TLR4-treated control fractures. (B) Histomorphometry showed that fractures in the AntagomiR-4485-3p-treated mice were composed of a markedly high proportion of bone, and low proportion of cartilage. In contrast, fractures in the siRNA-TLR4-treated mice were composed of low bone density and high cartilage density. (C-D) siRNA-TLR4-treated fractures in mice had increased callus area and decreased bone area compared with AntagomiR-4485-3p-treated mice. *p<0.05, **p<0.01, ***p<0.001.