Upregulation of microRNA-23b-3p induced by farnesoid X receptor regulates the proliferation and apoptosis of osteosarcoma cells

Abstract

Background: The downstream targets of farnesoid X receptor (FXR) such as miRNAs have a potent effect on the progression of many types of cancer. We aim to study the effects of FXR on osteosarcoma (OS) development and the potential role of microRNA-23b-3p.

Methods: The expressions of FXR and miR-23b-3p in normal osteoblasts and five osteosarcoma cell lines were measured. Their correlations were analyzed by Pearson's test and verified by the introduction of FXR agonist, GW4064. TargetScan predicted that cyclin G1 (CCNG1) was a target for miR-23b-3p. The transfection of FXR siRNA was performed to confirm the correlation between FXR and miR-23b-3p. We further transfected miR-23b-3p inhibitor into MG-63 cells, and the transfected cells were treated with 5 μM GW4064 for 48 h. Quantitative PCR (qPCR) and Western blot were performed for expression analysis. Cell proliferation, cell apoptosis rate, and cell cycle distribution were assessed by clone formation assay and flow cytometry.

Results: Scatter plot showed a positive correlation between FXR and miR-23b-3p (Pearson's coefficient test R² = 1.00, P = 0.0028). As CCNG1 is a target for miR-23b-3p, the treatment of GW4064 induced the downregulation of CCNG1 through upregulating miR-23b-3p. The inhibition of miR-23b-3p obviously promoted cell viability, proliferation, and cell cycle progression but reduced apoptosis rate of MG-63 cells; however, the treatment of GW4064 could partially reverse the effects of the inhibition of miR-23b-3p on OS cells.

Conclusions: Upregulated FXR by GW4064 can obviously suppress OS cell development, and the suppressive effects may rely on miR-23b-3p/CCNG1 pathway.

Keywords: Clone formation assay; Cyclin G1; Farnesoid X receptor; Osteosarcoma.

Fig. 1

Correlation of farnesoid X receptor (FXR) and microRNA-23b-3p expression in osteosarcoma (OS) cells and miR-23b-3p specifically targets cyclin G1 (CCNG1). As FXR might regulate the expression of miR-23b-3p in OS cells, we measured the expressions of FXR and miR-23b-3p in normal osteoblasts (hFOB1.19) and five osteosarcoma cell lines (MG-63, HOS, U2OS, SAOS2, SJSA1). a The relative expression levels of FXR were much downregulated in OS cell lines, especially in MG-63 cells. b The expressions of miR-23b-3p were obviously decreased, especially in MG-63 cells compared with hFOB1.19 cells. c Scatter plots showed the correlation of FXR and miR-23b-3p expression in normal osteoblasts and five osteosarcoma cell lines (Pearson’s coefficient test R² = 1.00, P = 0.0028). d The changes of miR-23b-3p levels were measured under different concentrations of the FXR agonist, GE4064 (0, 0.5, and 5 μM). e TargetScan predicted that the fragment of CCNG1-3′-UTR contained a binding site of miR-23b-3p. f The correlation between miR-23b-3p and CCNG1 was verified by luciferase reporter assay. Each value represents mean ± SEM (n = 3). GAPDH and U6 served as internal controls for cellular genes and miR-23b-3p, respectively. **p < 0.01 vs. hFOB1.19 cells or blank group; ^##p < 0.01 vs. 0.5 μM GW4064 group

Fig. 2

GW4064 treatment indirectly regulated cyclin G1 (CCNG1) expression via microRNA-23b-3p. CCNG1 was identified as a promising target of miR-23b-3p. Considering the positive correlation between farnesoid X receptor (FXR) and miR-23b-3p, we further assessed the correlation between FXR and CCNG1 expression. a The changes of CCNG1 mRNA and protein levels were measured by quantitative PCR (qPCR) and Western blot under the effects of increased concentrations of GW4064. b The transfection efficiency of FXR siRNA (siFXR) was measured by qPCR and Western blot. c The combined effect of siFXR and GW4064 on the expression of miR-23b-3p was examined by qPCR. d The combined effect of siFXR and GW4064 on CCNG1 expressions was measured by qPCR and Western blot. e We further transfected miR-23b-3p inhibitor and inhibitor control into MG-63 cells. The single and combined effects of inhibiting miR-23b-3p and GW4064 effects on CCNG1 were detected by qPCR and Western blot. f The changes of MG-63 cell viability were determined by Cell Counting Kit-8 (CCK-8) assay. Each value represents mean ± SEM (n = 3). GAPDH and U6 served as internal controls for cellular genes and miR-23b-3p, respectively. **p < 0.01 vs. blank (siNC or IC) group; ^##p < 0.01 vs. siNC + GW4064 group; ^p < 0.05, ^^p < 0.01 vs. I group

Fig. 3

GW4064 treatment inhibited cell proliferation and induced apoptosis and cell cycle arrest in MG-63 cells. We further study the effects of farnesoid X receptor (FXR) regulation on the development of MG-63 cells and the role of microRNA-23b-3p during the progress. a, b In order to determine the effects of FXR regulation on osteosarcoma (OS) cell proliferation, clone formation assay of MG-63 cells were performed under the single and combined effects of miR-23b-3p inhibition and GW4064 treatment. c, d The changes of apoptosis rate were analyzed by flow cytometry. e, f The cell cycle distribution of MG-63 cells was determined by flow cytometry. g, h The protein levels of several apoptotic genes (B-cell lymphoma-2 (Bcl-2), Bax, and Cleaved Caspase-3 (C caspase-3)) were analyzed by Western blot to further assess the effects of FXR regulation on OS cell apoptosis. Each value represents mean ± SEM (n = 3). GAPDH served as an internal control. *p < 0.05, **p < 0.01 vs. IC group; ^##p < 0.01 vs. IC + GW4064 group; ^^p < 0.01 vs. I group