MiRNA-34a inhibits human osteosarcoma proliferation by regulating the expression of ether-à-go-go 1 gene: a study in vitro

Abstract

Objective To detect the influence of miRNA-34a (miR-34a) on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation. Methods The osteoblastic cell line MG-63 and Saos-2, human osteoblastic cell line hFOB 1.19, 10 osteosarcoma tissues and 10 normal bone tissues were selected. The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction (qPCR). Next, a eukaryotic expression vector named pcDNA/miR-34a was constructed. Then, osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8, colony formation and xenograft model of nude mice. Finally, Western blot analysis was used to detect the expression of ether-a-go-go 1 (Eag1) gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2'-O-Methyl antisense oligoribonucleotide (miR-34a-2'-O-Me). Results Compared with normal bone tissues and osteoblastic cell line, miR-34a was down-regulated in osteosarcoma cell lines and tissues. Compared with the blank group and the control group, the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h: blank group (40.05±4.82)%, control group (36.88±4.66)%, miRNA-34a group (26.24±6.22)%; MG-63 96 h: blank group (83.55±5.95)%, control group (80.13±4.48)%, miRNA-34a group (30.21±7.26)%; Saos-2 72 h: blank group (46.45±8.15)%, control group (43.33±6.89)%, miRNA-34a group (26.81±3.17)%; Saos-2 96 h: blank group (84.79±4.10)%, control group (80.14±3.11)%, miRNA-34a group (31.77±5.17)%]. The similar results were obtained from colony formation assay (MG-63: blank group 83.40±3.29, control group 80.00±3.06, miR-34a group 24.40±2.71; Saos-2: blank group 85.00±3.32, control group 80.60±3.29, miR-34a group 30.40±4.94). The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment (all P < 0.001). Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1(P < 0.001). Conclusion MiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1. Moreover, it may be a novel target for osteosarcoma therapy. Key words: Osteosarcoma; MicroRNAs; Cell proliferation; Mice, nude; Gene, ether-a-go-go 1