Let-7a Inhibits Proliferation,Migration And Invasion Of Human Osteosarcoma Cells By Targeting Aurora-B In Vitro

Objective:To investigate let-7a inhibits proliferation,migration and invasion of human osteosarcoma cells by targeting Aurora-B in Vitro Methods:1.Twenty-one cases of osteosarcoma and adjacent tissues were collected and real-time quantitative PCR was used to detect the expression level of let-7a in osteosarcoma and paracancerous tissues,osteosarcoma cells(143B cells and U2-OS cells)and hFOB1.19 cells.2.The OS cells were treated with let-7a mimic and negative mimic.The ability of cells proliferation,migration and invasion was measured using CCK8,wound healing and transwell invasion assays.3.The bioinformatics database predicts that the target gene of Let-7a is Aurora-B.dual-luciferase assay to verify whether Aurora-B is a direct target gene for let-7a.Real-time quantitative PCR was used to detect the difference in expression levels of let-7a between osteosarcoma and normal tissues,osteosarcoma cells(143B cells and U2-OS cells)and hFOB1.19 cells.Finally,the expression relationship between let-7a and Aurora-B was analyzed in osteosarcoma tissues.4.In the rescue experiment,the OS cells were treated with Lv-Aurora-B combined with let-7a mimic.The ability of cells proliferation,migration and invasion was measured using CCK8,wound healing and transwell invasion assays.The protein of Aurora-B,NF-κβ,MMP2 and MMP9 was measured by western blot.Results:1.The results of real-time quantitative PCR showed that the expression of let-7a in osteosarcoma was significantly lower than that in adjacent tissues,and the expression of let-7a in osteosarcoma 143 B and U2-OS cells was also significantly lower than that of hFOB1.19 cells.(P<0.05).2.The results showed that the viability,migratory and invasive ability weresignificant lower in cells treated with let-7a mimic than those cells treated with negative mimic.(P<0.05);3.It was predicted by targetscan database and starBase database that let-7a can be paired with seed sequence of Aurora-B mRNA 3’ UTR.dual-luciferase assay showed that let-7a recognized the 3’ UTR of Aurora-B mRNA constructed into the luciferase reporter vector,thereby inhibiting luciferase activity(P < 0.05).And there was no significant difference in the 3’ UTR of the mutated Aurora-B mRNA.4.Real-time quantitative PCR showed that the expression of Aurora-B in osteosarcoma tissues was significantly higher than that in adjacent tissues,and the expression of Aurora-B in 143 B cells and U2-OS cells were also significantly higher than that of hFOB1.19 cells(P<0.05).The Pearson correlation assay showed that there was a negative relationship between let-7a and Aurora-B in osteosarcoma tissues(r=-0.7575,P<0.05).5.The results showed the tumor inhibition of let-7a was partly rescued by up-regulation of Aurora-B in OS cells.Western blot analysis showed that the expression levels of Aurora-B,NF-κβ,MMP2 and MMP9 proteins in 143 B cells and U2-OS cells transfected with let-7a inhibitor were elevated.And the expression levels of Aurora-B,NF-κβ,MMP2 and MMP9 proteins in the let-7a mimic+LV-Aurora-B co-transfection group were partially restored,compared with the let-7a mimic group.(P<0.05)Conclusion:Let-7a inhibits OS cell malignant phenotype by targeting Aurora-B at least partially.Targeting of let-7a and Aurora-B/NF-κβ may be a novel therapeutic strategy for treatment OS.