Long noncoding RNA OR3A4 promotes the proliferation and invasion of osteosarcoma cells by sponging miR-1227-5p

Abstract

Background: Long noncoding RNAs (lncRNAs) have been identified as key players in promoting tumourigenesis in osteosarcoma. LncRNA OR3A4 (OR3A4) has been reported as an oncogene in a number of tumours. However, the clinical value of OR3A4 in osteosarcoma and the role of OR3A4 in osteosarcoma progression are still unknown.

Methods: The expression levels of OR3A4 in the tumour tissue of osteosarcoma patients and osteosarcoma cell lines were detected by RT-PCR. Kaplan-Meier analysis and log-rank test were performed to evaluate the relationship between the level of OR3A4 expression and the prognosis of osteosarcoma patients. We investigated the association between the tissue expression levels of OR3A4 and different clinicopathological characteristics of osteosarcoma patients by χ² tests. Bioinformatic databases and luciferase reporter assays were used to predict and validate the target microRNA of OR3A4. Finally, the role of OR3A4 in the proliferation and invasion of osteosarcoma cells was tested by MTT and Transwell assays, respectively.

Results: We observed that the expression level of OR3A4 was upregulated in the tumour tissue of osteosarcoma patients (p < 0.001) and osteosarcoma cell lines (p < 0.01) compared with the normal adjacent tissue and a normal human foetal osteoblastic cell line, respectively. The survival curve revealed that patients with high expression levels of OR3A4 had lower overall survival. Increased OR3A4 expression in osteosarcoma patients was associated with distant metastasis (p = 0.02) and advanced clinical stage (p < 0.001). In addition, bioinformatics analysis and luciferase reporter assays verified the complementary binding between OR3A4 and miR-1227-5p. Furthermore, we found that OR3A4 acted as a miR-1227-5p "sponge" to modulate osteosarcoma cell proliferation and invasion via downregulation of miR-1227-5p.

Conclusion: OR3A4 promotes osteosarcoma cell proliferation and invasion by sponging miR-1227-5p, which might be related to the metastasis of osteosarcoma and could be used as a potential prognostic biomarker and therapeutic target in osteosarcoma.

Keywords: OR3A4; Osteosarcoma invasion; Prognosis; miR-1227-5p.

Fig. 1

OR3A4 was upregulated in osteosarcoma tissue and cell lines, which was correlated with poor prognosis. (A) The expression of OR3A4 in osteosarcoma patient tissue was significantly higher than that in normal adjacent tissue. (B) Higher expression of OR3A4 was found in osteosarcoma cell lines (U2OS, HOS and MG-63) than in the control cell line (hFOB). (C) Kaplan–Meier analysis showed that patients with high OR3A4 expression had significantly lower survival than patients with low OR3A4 expression. **p < 0.01, ***p < 0.001, compared to the control group.

Fig. 2

Downregulation of OR3A4 inhibited the proliferation and invasion of osteosarcoma cells. (A) qRT-PCR was used to detect the expression of OR3A4 in the MG-63 cell line transfected with si-OR3A4. (B) MTT assay was performed to analyse the effect of OR3A4 on MG-63 cell proliferation. (C) Transwell assay was conducted to determine the effect of OR3A4 on MG-63 cell invasion **p < 0.01 compared to the control group.

Fig. 3

MiR-1227-5p was the potential target microRNA of OR3A4, which was predicted and validated by bioinformatics analysis and luciferase reporter assay. (A) A flow chart to screen the miRNAs based on the diagrams. (B) The complementary binding site of OR3A4 and miR-1227-5p was predicted by bioinformatics. (C) qRT-PCR was performed to observe the expression of miR-1227-5p in MG-63 cells transfected with si-OR3A4. (D) Luciferase reporter assay was used to confirm the predicted binding. (E) The expression of miR-1227-5p in clinical stages I/II and III/IV of osteosarcoma was detected by qRT-PCR. **p < 0.01, ***p < 0.001, compared to the control group.

Fig. 4

OR3A4 accelerated osteosarcoma cell proliferation and invasion by targeting miR-1227-5p. (A) The expression levels of miR-1227-5p in MG-63 cells transfected with si-OR3A4 and miR-1227-5p inhibitor. (B) MTT assay was used to observe the role of si-OR3A4+miR-1227-5p inhibitor on MG-63 cell proliferation. (C) Transwell assay was used to detect the effect of si-OR3A4+miR-1227-5p inhibitor on MG-63 cell invasion. **p < 0.01 compared to the control group.