MicroRNA-17 promotes osteosarcoma cells proliferation and migration and inhibits apoptosis by regulating SASH1 expression

https://doi.org/10.1016/j.prp.2018.10.012Get rights and content

Abstract

MicroRNAs (miRNAs) are abnormally expressed in numerous diseases, which are intimately associated with cell proliferation, migration and invasion. Recent study indicated that miR-17 may be involved in regulating osteosarcoma (OS) occurrence and development, but its function and mechanism have not been reported. In this study, quantitative real-time PCR (qRT-PCR) was used to measure the expression of miR-17, and Western blotting assay was performed to measure the expressions of SAM and SH3 domain containing 1 (SASH1), phosphoinoinositide-3 kinase (PI3K), protein kinase B (AKT), Caspase3, Bcl-2 gene family (Bcl-2, Bax) and matrix metalloprotein (MMP-2, MMP-9) in MG-63 cells. Luciferase reporter assay was conducted to confirm the target of SASH1 by miR-17. Cell proliferation, migration, invasion and apoptosis assay was performed to investigate the role of miR-17 in OS cells. We found that the expression of miR-17 was significantly up-regulated in OS cell lines. MiR-17 inhibitor inhibited the proliferation ability, and induced apoptosis of OS cells. Besides, miR-17 inhibitor prevented the migration and invasion of OS cells. Further, we identified that SASH1 was a target gene of miR-17. In addition, knockdown of miR-17 increased the protein expression of SASH1, and regulate related genes of cell proliferation, invasion and anti-apoptosis in the downstream of OS cells. These findings indicated that miR-17 was over-expressed and promoted cell proliferation, migration and inhibited cell apoptosis by targeting SASH1 in OS cells.

Section snippets

Background

Osteosarcoma is the most common primary malignancy tumors of bone in children and young adults [1]. Despite advances in new treatment such as chemotherapy, surgery, and combination of chemotherapy, the rate of 5-year survival rises to 60%–70%, and the rate of metastasis or recurrence is significantly decreased to approximately 30% [[2], [3], [4]]. Previous studies reported a lack of complete understanding with regard to the initiation and development of OS metastasis. The underlying molecular

Cell culture and transfection

Human osteosarcoma cell lines U2OS and MG-63 were cultured in Dulbecco's Modified Eagle's Medium (DMEM; SH30022.01, Hyclone, GE Health Care, USA), and Saos-2 cell lines were cultured in RPMI 1640 mediums (Gibco; SH30096.01, Hyclone, GE Health Care, USA). Culture medium was supplemented with 10% foetal bovine serum (FBS; Gibco; SV30087.02, Thermo Fisher Scientific, Waltham, USA), 100 mg/ml streptomycin, and 100 IU/ml penicillin in a 5% CO2/water-saturated incubator at 37 °C.

Cell transfection

The MG-63 cells were

MiR-17 was significantly up-regulated in OS cell lines

The expression levels of miR-17 in human OS cell lines (MG63, Saos-2, U2OS) and the normal human osteoblast cell line hFoB1.19 were analyzed using qRT-PCR analysis. As shown in Fig. 1A, the expression of miR-17 in MG-63, Saos-2 and U2OS were significantly increased compared with hFoB1.19, and MG-63 cells had the highest expression of miR-17. These results indicated that miR-17 was significantly up-regulated in OS cell lines. And we used MG-63 cells to perform further analysis.

MiR-17 promoted cell proliferation, migration and inhibited apoptosis in OS cells

To explore the

Discussion

OS are aggressive primary tumor of the bone. The etiology and molecular pathogenesis of OS remain unclear. Thus, it is urgent to explore the molecular mechanisms governing rapidly growth of OS [15]. MiRNAs are important epigenetic regulators of gene expression at the posttranscriptional level. Several miRNAs have recently been found to be involved in basic biological processes, including cell proliferation, differentiation and apoptosis. Several miRNAs have been linked to OS, however, their

Conclusion

In summary, our study detected a high expression level of miR-17 in OS cell, which was functioned as an oncogenic miRNA by promoting OS cell proliferation, migration and invasion. This study provided important evidences in OS development. And this study suggested that targeting miR-17/SASH1 axis might represent a potential therapeutic strategy to eradicate OS cells.

Authors' contributions

DW and HZ was responsible for the study design and the acquisition of data, undertook data analysis and performed the functional experiments. FJ helped to design the experiments and interpret the data. WD undertook project design and manuscript revisions.

Ethical approval and consent to participate

Not applicable

Consent for publication

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (No: 81272942, No: 81702666 and No: 81502328)

Competing interests

The authors declare that they no financial conflicts of interest

Availability of data and materials

The datasets used/or analyzed during the current study are available from the corresponding author on reasonable request

Acknowledgements

Not applicable

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