Abstract:

Objective To investigate the effects of miR-219a-5p on proliferation, invasion and migration of osteosarcoma U2OS cells by regulating high mobility group A2 （HMGA2）. Methods Real-time quantitative PCR was used to detect miR-219a-5p mRNA expression levels in osteosarcoma U2OS cells and normal osteoblasts hFOB1.19. The U2OS cells were transfected with miR-219a-5p mimic （miR-219a-5p mimic group） and negative control mimic （mimic NC group） by liposome transfection. The expression levels of miR-219a-5p and HMGA2 mRNA in transfected cells were detected by real-time quantitative PCR. The level of HMGA2 protein was detected by Western blotting, cell proliferation ability was detected by CCK-8 assay and clonogenesis assay, cell migration ability was detected by scratching assay, cell invasion ability was detected by Transwell chamber assay, and the relationship between miR-219a-5p and HMGA2 was verified by double luciferase reporter gene assay. Results Real-time quantitative PCR showed that the expression level of miR-219a-5p in osteosarcoma U2OS cells （0.11±0.01） was significantly lower than that in normal osteoblasts （1.00±0.06）, with a statistically significant difference （t=26.83, P<0.001）. The results of CCK-8 showed that the cell absorbance values of the mimic NC group and miR-219a-5p mimic group were 0.52±0.02 and 0.42±0.02 after 24 h, 0.85±0.03 and 0.60±0.03 after 48 h, and 1.12±0.02 and 0.72±0.02 after 72 h respectively. The proliferation activity of the miR-219a-5p mimic group was significantly lower than that of the mimic NC group, with statistically significant differences （t=6.97, P<0.001; t=16.65, P<0.001; t=26.78, P<0.001）. The results of clonogenesis assay showed that the number of clones in the miR-219a-5p mimic group was 157.00±15.39, which was significantly lower than that in the mimic NC group （294.00±15.51）, with a statistically significant difference （t=9.70, P<0.001）. The results of scratch experiment showed that the percentage of scratch area in the miR-219a-5p mimic group was （40.53±2.92）% after 24 h culture, which was significantly higher than that in the mimic NC group [（21.71±3.11）%], with a statistically significant difference （t=7.26, P=0.002）. The results of Transwell chamber assay showed that the number of cells penetrating the membrane in the miR-219a-5p mimic group was 128.67±18.67, which was significantly lower than that in the mimic NC group （317.67±14.33）, with a statistically significant difference （t=15.65, P<0.001）. The results of double luciferase reporter gene assay showed that in MUT-HMGA2 cells, transfection with miR-219a-5p mimic （4.30±0.26） had no significant effect on luciferase activity compared with the mimic NC group （4.40±0.28）, with a statistically significant difference （t=0.85, P=0.690）. In WT-HMGA2 cells, compared with the mimic NC group （4.50±0.25）, the lucifase activity of the miR-219a-5p mimic group （2.88±0.16） was significantly decreased, with a statistically significant difference （t=19.15, P<0.001）. After miR-219a-5p was overexpressed, HMGA2 mRNA and protein expressions in osteosarcoma U2OS cells （0.77±0.01; 0.37±0.01） were downregulated compared with the mimic NC group （1.00±0.02; 1.00±0.01）, with statistically significant differences （t=16.38, P<0.001; t=42.02, P<0.001）. Conclusion In osteosarcoma cells, miR-219a-5p can inhibit the proliferation, migration and invasion of osteosarcoma cells by down regulating the expression of HMGA2.

Key words: Osteosarcoma, MicroRNAs, HMGA2 protein, Cell proliferation