MiR-490-3p promotes cell apoptosis and cell-cycle arrest in osteosarcoma via the modulation of CDCA8/ATF3 by targeting NUSAP1

Abstract

Background: Micro RNA-490-3p (miR-490-3p) is associated with a variety of malignancies. However, the role of miR-490-3p in osteosarcoma and its underlying mechanism are not yet fully understood. This study aimed to explore the role and the mechanism of miR-490-3p in osteosarcoma.

Methods: MiR-490-3p and nucleolar and spindle-associated protein 1 (NUSAP1) expression in osteosarcoma was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), wound-healing, and transwell assays were used to detect cell proliferation, migration and invasion. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot were used to evaluate the cell apoptosis. Flow cytometry was used to assess the cell cycle. In addition, luciferase reporter assay was used to confirm the binding of miR-490-3p and NUSAP1. Western blot and RT-qPCR was used to examine cell division cycle associated 8 (CDCA8) and activating transcription factor 3 (ATF3) expression.

Results: MiR-490-3p expression was significantly decreased in the osteosarcoma cells. Following the transfection with miR-490-3p mimic, it was found that 143B cell proliferation, migration, and invasion were inhibited, while the cell apoptotic levels and cell-cycle arrest were promoted, accompanied with decreased B cell lymphoma protein-2 (Bcl-2) protein expression, and increased protein expressions of Bcl-2-associated X (Bax), cleaved caspase-3, and cleaved caspase-9. In addition, miR-490-3p was found to bind to NUSAP1, and negatively regulate NUSAP1 expression. NUSAP1 upregulation reversed the inhibitory effects of miR-490-3p overexpression on cell proliferation, migration, and invasion, and the promoting effects on cell apoptosis and cell-cycle arrest in osteosarcoma. Moreover, miR-490-3p was identified to mediate CDCA8/ATF3 by targeting NUSAP1.

Conclusions: MiR-490-3p upregulation inhibited cell proliferation and metastasis but promoted the cell apoptosis and cell-cycle arrest in osteosarcoma via the regulation of CDCA8/ATF3 by targeting NUSAP1. Thus, miR-490-3p might be a potential therapeutic target for the treatment of osteosarcoma.

Keywords: Micro RNA-490-3p (miR-490-3p); apoptosis; cell-cycle arrest; nucleolar and spindle-associated protein 1 (NUSAP1); osteosarcoma.

Figure 1

MiR-490-3p overexpression decreases the proliferation, migration and invasion of osteosarcoma cells. (A) RT-qPCR was used to analyze miR-490-3p expression in osteosarcoma and hFOB1.19 cell lines; (B) RT-qPCR was used to analyze miR-490-3p mRNA expression; (C) CCK-8 assays were used to analyze cell proliferation; (D,E) wound-healing assays were used to analyze cell migration. The cells were observed using a light microscope. (F,G) transwell assays were used to analyze cell invasion. The cells were observed using a light microscope. Cells were exposed to 0.1% crystal violet staining. **, P<0.01; ***, P<0.001. miR-490-3p, micro RNA-490-3p; RT-qPCR, real-time quantitative polymerase chain reaction; CCK-8, Cell Counting Kit-8; mRNA, messenger RNA; NC, negative control; ns, no significance.

Figure 2

MiR-490-3p overexpression facilitates the cell apoptotic level and cycle arrest in osteosarcoma. (A,B) TUNEL was used to assess cell apoptosis and DAPI was used for staining; (C) Western blot was used to examine the proteins associated with apoptosis; (D,E) flow cytometry was used to examine the cell cycle. ***, P<0.001. miR-490-3p, micro RNA-490-3p; TUNEL, transferase dUTP nick end labelling; NC, negative control; DAPI, 4’,6-diamidino-2-phenylindole; ns, no significance.

Figure 3

MiR-490-3p binds to NUSAP1. (A) The binding of miR-490-3p with NUSAP1; (B) luciferase reporter assays were used to confirm the luciferase activity; (C) Western blot and RT-qPCR were used to analyze NUSAP1 mRNA and protein expression. ***, P<0.001. miR-490-3p, micro RNA-490-3p; RT-qPCR, real-time quantitative polymerase chain reaction; mRNA, messenger RNA; NC, negative control; ns, no significance.

Figure 4

NUSAP1 overexpression reverses the effects of miR-490-3p on the proliferation, migration and invasion of osteosarcoma cells. (A) RT-qPCR was used to analyze NUSAP1 mRNA expression in 143B cells transfected with Over-NUSAP1; (B) CCK-8 assays were used to analyze cell proliferation; (C,D) wound-healing assays were used to analyze cell migration. The cells were observed using a light microscope. (E,F) transwell assays were used to analyze cell invasion. The cells were observed using a light microscope. Cells were exposed to 0.1% crystal violet staining; ***, P<0.001. miR-490-3p, micro RNA-490-3p; RT-qPCR, real-time quantitative polymerase chain reaction; CCK-8, Cell Counting Kit-8; mRNA, messenger RNA; NC, negative control; ns, no significance.

Figure 5

NUSAP1 overexpression reverses the effects of miR-490-3p on 143B cell apoptosis and cell-cycle arrest. (A,B) TUNEL was used to examine cell apoptosis and DAPI was used for staining; (C,D) flow cytometry was used to examine cell cycle; (E) Western blot was used to examine apoptosis-related proteins. **, P<0.01, ***, P<0.001. miR-490-3p, micro RNA-490-3p; TUNEL, transferase dUTP nick end labelling; NC, negative control; DAPI, 4’,6-diamidino-2-phenylindole; ns, no significance.

Figure 6

MiR-490-3p mediates CDCA8/ATF3 by targeting NUSAP1. (A) According to the STRING database, NUSAP1 binds to CDCA8; (B) western blot was used to detect the protein expression of the CDCA8 and ATF3. **, P<0.01; ***, P<0.001. miR-490-3p, micro RNA-490-3p; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; NC, negative control; ns, no significance.