Role of microRNA-92a in metastasis of osteosarcoma cells in vivo and in vitro by inhibiting expression of TCF21 with the transmission of bone marrow derived mesenchymal stem cells

Abstract

Background: This study aims to investigate the role of microRNA-92a (miR-92a) in metastasis of osteosarcoma (OS) cells in vivo and in vitro by regulatingTCF21 with the transmission of bone marrow derived mesenchymal stem cells (BMSCs).

Methods: BMSCs were isolated, purified and cultured from healthy adult bone marrow tissues. The successfully identified BMSCs were co-cultured with OS cells, and the effects of BMSCs on the growth metastasis of OS cells in vitro and in vivo were determined. qRT-PCR and western blot analysis was used to detect the expression of miR-92a and TCF21 in OS cells and OS cells co-cultured with BMSCs. Proliferation, invasion and migration of OS cells transfected with miR-92a mimics and miR-92a inhibitors was determined, and the tumorigenesis and metastasis of OS cells in nude mice were observed. Expression of miR-92a and TCF21 mRNA in tissue specimens as well as the relationship between the expression of miR-92a and the clinicopathological features of OS was analyzed.

Results: BMSCs promoted proliferation, invasion and migration of OS cells in vitro together with promoted the growth and metastasis of OS cells in vivo. Besides, high expression of miR-92a was found in OS cells co-cultured with BMSCs. Meanwhile, overexpression of miR-92a promoted proliferation, invasion and migration of OS cells in vitro as well as promoted growth and metastasis of OS cells in vivo. The expression of miR-92a increased significantly, and the expression of TCF21 mRNA and protein decreased significantly in OS tissues. Expression of miR-92a was related to Ennecking staging and distant metastasis in OS patients.

Conclusion: Collectively, this study demonstrates that the expression of miR-92a is high in OS and BMSCs transfers miR-92a to inhibit TCF21 and promotes growth and metastasis of OS in vitro and in vivo.

Keywords: Invasion; MicroRNA-92a; Migration; Osteosarcoma; Proliferation; TCF21.

Fig. 1

Identification of BMSCs. a Observation of cell morphology by an inverted microscope (×100); b identification of cell surface markers by flow cytometry; c identification of osteogenic differentiation ability of cells by alizarin red staining (×40); d identification of adipogenic differentiation ability of cells by oil red O staining (×40). The experiment was repeated three times

Fig. 2

BMSCs promote proliferation of OS cells in vitro. Repetitions = 3; the data was analyzed by the t test; *, P < 0.05 vs. 143B cells or SaOS2 cells

Fig. 3

BMSCs promote the invasion and migration of OS cells in vitro. a Transwell assay detection of the number of cell invasion in each group (× 100); b detection of cell migration in each group by scratch test. Repetitions = 3; the data was analyzed by the t test

Fig. 4

BMSCs promote the growth and metastasis of OS cells in vivo. a Tumor growth curve of two groups of nude mice; b tumor weight in two groups of nude mice; c lung metastasis in two groups of nude mice; d observation of lung metastasis in two groups of nude mice by HE staining (× 200); e number of lung metastatic nodules in two groups of nude mice; N = 6; the data was analyzed by the t test; *, P < 0.05 vs. 143B cells

Fig. 5

High expression of miR-92a in OS cells co-cultured with BMSCs. a Detection of miR-92a expression in 143B and SaOS2 cells before and after co-culture by qRT-PCR; b expression of miR-92a in 143B and SaOS2 cells detected by qRT-PCR; Repetitions = 3; the data was analyzed by the t test or one-way ANOVA; After ANOVA analysis, the LSD-t was used for pairwise comparison; *P < 0.05 vs. the blank group

Fig. 6

Overexpression of miR-92a promotes proliferation of OS cells in vitro. Repetitions = 3; the data was analyzed by the t test or one-way ANOVA; After ANOVA analysis, the LSD-t was used for pairwise comparison; *P < 0.05 vs. the blank group

Fig. 7

Overexpression of miR-92a promotes invasion and migration of OS cells. a Detection of the number of invasive cells in each group by Transwell assay (× 100); b detection of cell migration in each group by scratch test; Repetitions = 3; the data was analyzed by one-way ANOVA; After ANOVA analysis, the LSD-t was used for pairwise comparison; *P < 0.05 vs. the blank group

Fig. 8

Down-regulation of miR-92a inhibit the growth and metastasis of OS cells in vivo. a Tumor growth curve of nude mice in each group; b tumor weight of nude mice in each group; c lung metastasis of nude mice in each group; d observation of lung metastasis in nude mice by HE staining (× 200); e the number of lung metastatic nodules in nude mice in each group; N = 6; the data was analyzed by one-way ANOVA; After ANOVA analysis, the LSD-t was used for pairwise comparison; *P < 0.05 vs. the blank group

Fig. 9

TCF21 is suggested to be a direct target gene of miR-92a. a Detection of TCF21 mRNA levels in OS cells before and after co-culture with BMSCs by qRT-PCR; b detection of TCF21 protein expression in OS cells before and after co-culture with BMSCs by western blot analysis; c detection of TCF21 mRNA expression in OS cells of each group by qRT-PCR; d detection of TCF21 protein expression in OS cells by western blot analysis; e online prediction of targeting relationship between miR-92a and TCF21; f identification of targeting relationship between miR-92a and TCF21 by luciferase activity determination; Repetitions = 3; the data was analyzed by the t test or one-way ANOVA; After ANOVA analysis, the LSD-t was used for pairwise comparison; *P < 0.05 vs. the blank or the mimics NC group

Fig. 10

Expression of miR-92a and TCF21 in OS tissues and osteochondroma tissues. a In situ hybridization used to detect the expression of miR-92a in tissues (×200); b in situ hybridization used to detect the expression of TCF21 in tissues (×200); c immunohistochemical detection of TCF21 protein expression in tissues (×200)