Circ-0003998 promotes cell proliferative ability and invasiveness by binding to miR-197-3p in osteosarcoma

L. Wang, Z.-G. Du, H. Huang, F.-S. Li, G.-S. Li, S.-N. Xu

Department of Bone and Soft Tissue Oncology, Liaoning Provincial People’s Hospital, Shenyang, China. wlaimy@163.com

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• Oncology

OBJECTIVE: The aim of this study was to investigate the level of circ-0003998 in osteosarcoma tissues and cell lines, and to analyze its relation with prognosis of patients, as well as its effect on biological behaviors of osteosarcoma cells. In addition, the potential mechanism of circ-0003998 in promoting osteosarcoma cell proliferation and invasion was explored.
PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine circ-0003998 expression in 60 clinical osteosarcoma tissues and cell lines. The association between circ-0003998 expression and the overall survival rate of patients was explored. After shRNA-circ-0003998 was constructed to down-regulate circ-0003998 expression in osteosarcoma cell lines, the proliferation of osteosarcoma cells was observed through the Cell Counting Kit-8 (CCK-8) and colony formation assay. Meanwhile, cell invasiveness was detected by the transwell invasion assay. Bioinformatics was used to search for microRNAs (miRNAs) that contained the direct effect on circ-0003998. Subsequently, the luciferase reporter vector of circ-0003998 or Krüppel-like factor 10 (KLF10) containing miR-197-3p binding site was constructed. Then, the binding of circ-0003998 or KLF10 to miR-197 was detected using the Dual-Luciferase assay. Furthermore, the function recovery experiment was designed to validate the biological function of circ-0003998 and miR-197 in osteosarcoma.
RESULTS: Compared to normal control tissues and cells, the expression of circ-0003998 was significantly up-regulated in both osteosarcoma tissue samples and cell lines. Highly-expressed circ-0003998 was significantly associated with poor overall survival of patients with osteosarcoma. In vitro experiments revealed that the down-regulation of circ-0003998 significantly inhibited the proliferative ability and invasiveness of osteosarcoma cells. Bioinformatics analysis and Dual-Luciferase reporter gene assay indicated that circ-0003998 might bind to miR-197-3p in MG-63, and Saos-2 cell lines. Meanwhile, the functional recovery experiment demonstrated that inhibiting miR-197-3p expression could partially restore the changes in cellular biological behaviors induced by circ-0003998 down-regulation in MG-63 and Saos-2 cells. In addition, miR-197-3p was remarkably down-regulated in osteosarcoma tissues, while KLF10 was up-regulated. However, KLF10 was significantly up-regulated after the knockdown of miR-197-3p in osteosarcoma cells.
CONCLUSIONS: Circ-0003998 plays a vital role in promoting the development of osteosarcoma, whose high expression can predict poor clinical prognosis. Circ-0003998 is highly expressed in osteosarcoma tissues and cell lines. The down-regulation of its level can significantly inhibit the proliferative ability and invasiveness of osteosarcoma cells. Meanwhile, circ-0003998 up-regulates the expression of KLF10 by binding to miR-197-3p, thereby promoting osteosarcoma cell growth and invasion, and accelerating the progression of osteosarcoma.

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