MicroRNA-21 Inhibits the Apoptosis of Osteosarcoma Cell Line SAOS-2 via Targeting Caspase 8

Abstract

Currently, multiple microRNAs (miRNAs) have been found to play vital roles in the pathogenesis of osteosarcoma. This study aimed to investigate the role of miR-21 in osteosarcoma. The level of miR-21 in 20 pairs of osteosarcoma and corresponding adjacent tissues was monitored by qPCR. Human osteosarcoma cell line SAOS-2 was transfected with either miR-21 mimic or miR-21 inhibitor, and then cell viability, survival, and apoptosis were measured by MTT, colony formation assay, and flow cytometry. A target of miR-21 was predicted by the microRNA.org database and verified in vitro by using luciferase reporter, qPCR, and Western blot analyses. Finally, cells were cotransfected with siRNA against caspase 8 and miR-21 inhibitor, and the apoptotic cell rate was determined again. Results showed that the mRNA level of miR-21 was highly expressed in osteosarcoma tissues compared with adjacent tissues. Overexpression of miR-21 improved cell viability and survival but suppressed apoptosis. Caspase 8 was a direct target of miR-21, and it was negatively regulated by miR-21. Moreover, miR-21 suppression attenuated caspase 8 silencing and induced the decrease in apoptosis. In conclusion, overexpression of miR-21 suppressed SAOS-2 cell apoptosis via directly targeting caspase 8.

Figure 1

MicroRNA-21 (miR-21) was upregulated in osteosarcoma. mRNA level expression of miR-21 in osteosarcoma tissue and adjacent tissue samples (n = 20) was measured by quantitative PCR (qPCR). ***p < 0.001 when compared to the control.

Figure 2

Overexpression of miR-21 increased SAOS-2 cell survival while suppressing apoptosis. miR-21 mimic, miR-21 inhibitor, or control was transfected into SAOS-2 cells. Subsequently, (A) the transfection efficiency was tested by qPCR, (B) cell viability was determined by MTT assay, (C) cell survival was measured by colony formation assay, and (D, E) the apoptotic cell rate was detected by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to the control.

Figure 3

Caspase 8 was a target gene of miR-21. (A) The target of miR-21 was predicted using the microRNA.org database. (B) miR-21 mimic or (C) miR-21 inhibitor was cotransfected with the pmiR-Report vector containing full-length caspase 8 3′-UTR or a mutated seed site within pmiR-caspase 8 into SAOS-2 cells, and dual-luciferase reporter assay was then performed. NS, no significance. **p < 0.01, ***p < 0.001 when compared to the control.

Figure 4

Capase-8 was negatively regulated by miR-21. Cells were transfected with miR-21 mimic, miR-21 inhibitor, or control. Then (A) the mRNA level expression of caspase 8 was measured by qPCR, and (B, C) the protein expressions of caspase 8 and cleaved caspase 8 were measured by Western blot. Cells were transfected with small interfering RNA (siRNA) against caspase 8 and/or the miR-21 inhibitor, and then (D) the mRNA level of caspase 8 and (E, F) the protein expressions of caspase 8 and cleaved caspase 8 were measured. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to the control.

Figure 5

Overexpression of miR-21 suppressed SAOS-2 cell apoptosis via targeting caspase 8. (A, B) Cells were transfected with siRNA against caspase 8 and/or the miR-21 inhibitor, and then the apoptotic cell rate was determined by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to the control.