| ObjectiveIn this study,the effect of miR-27a-3p targeting FBXW7 on the biological behavior of osteosarcoma cells was studied through in vitro cell experiments,and the mechanism of action was explored,so as to lay an experimental foundation for molecular targeted therapy of osteosarcoma.Methods（1）miR-27a-3p expressed in osteosarcoma cells and its influence on osteosarcoma cell function:1.Expression of miR-27a-3p in osteosarcoma cells: The expression levels of miR-27a-3p in osteosarcoma cells MG-63,Saos-2 and normal osteoblasts h FOB1.19 were detected by RT-PCR assay.2.Effects of miR-27a-3p overexpression on proliferation,migration and apoptosis of osteosarcoma cells: miR-27a-3p mimic was transfected into osteosarcoma cells MG-63 and Saos-2,and the transfection efficiency was detected by RT-PCR.After the transfection,the proliferation,migration and apoptosis of MG-63 and Saos-2 cells were detected by CCK-8 assay,Transwell migration assay and flow cytometry.3.Effects of miR-27a-3p knockdown on proliferation,migration and apoptosis of osteosarcoma cells: miR-27a-3p inhibitor was transfected into osteosarcoma cells m G-63 and Saos-2,and the transfection efficiency was detected by RT-PCR assay.The proliferation,migration and apoptosis of MG-63 and Saos-2 cells were detected by CCK-8 assay,Transwell migration assay and flow cytometry.（2）Expression of FBXW7 in osteosarcoma cells and its effect on osteosarcoma cell function:1.Screening of miR-27a-3p target genes: the target genes of miR-27a-3p were queried and screened by Target Scans Human7.2,and the targeting relationship between miR-27a-3p and FBXW7 was verified by double luciferase reporter assay.2.Expression of FBXW7 in osteosarcoma cells: The expression levels of FBXW7 m RNA in osteosarcoma cells MG-63,Saos-2 and h FOB1.19 were detected by RT-PCR assay.3.Effects of overexpression of FBXW7 on proliferation and migration of osteosarcoma cells: Osteosarcoma cells MG-63 and Saos-2 were transfected with PCDNA3.1-Flag-FBXW7（an overexpressed plasmid of FBXW7）,and the transfection efficiency was detected by RT-PCR and Western blot.After transfection,the proliferation and migration of osteosarcoma cells MG-63 and Saos-2 were detected by CCK-8 assay and Transwell migration assay respectively.4.Effects of FBXW7 knockdown on osteosarcoma cell proliferation and migration: Osteosarcoma cells MG-63 and Saos-2 were transfected with si RNA-FBXW7,and the transfection efficiency was detected by RT-PCR and Western blot assay.After transfection,the proliferation and migration of OSTEosarcoma cells MG-63 and Saos-2 were detected by CCK-8 assay and Transwell migration assay respectively.（3）The role and mechanism of miR-27a-3p targeting FBXW7:1.miR-27a-3p targeted regulation of FBXW7 expression in osteosarcoma cells:Western blot was used to detect the expression of FBXW7 in osteosarcoma cells MG-63 and Saos-2 after transfection with miR-27a-3p mimic and miR-27a-3p inhibitor,respectively.2.miR-27a-3p regulates β-catenin expression in osteosarcoma cells: Western blot was used to detect the expression of β-catenin in osteosarcoma cells MG-63 and Saos-2 after transfection with miR-27a-3p mimic and miR-27a-3p inhibitor.3.miR-27a-3p regulates the proliferation and migration of osteosarcoma cells by targeting FBXW7 inhibition: After transfection of osteosarcoma cells MG-63 and Saos-2 with miR-27a-3p inhibitor and miR-27a-3p inhibitor+si FBXW7,respectively,the proliferation and migration of cells in each group were detected by CCK-8 assay and Transwell migration assay.Results（1）Compared with normal osteoblast h FOB1.19,miR-27a-3p expression level in osteosarcoma cells MG-63 and Saos-2 was increased（P<0.05）.Transfection with miR-27a-3p mimic increased the expression level of miR-27a-3p in osteosarcoma cells（P<0.05）.The proliferation and migration of osteosarcoma cells were increased（P<0.05）,and the apoptosis rate of osteosarcoma cells was decreased（P<0.05）.Transfection with miR-27a-3p inhibitor decreased the expression of miR-27a-3p in osteosarcoma cells（P<0.05）,decreased the proliferation and migration of osteosarcoma cells（P<0.05）,and increased the apoptosis rate of osteosarcoma cells（P<0.05）.（2）According to bioinformatics,FBXW7 was predicted to be one of the target genes of miR-27a-3p.The results of double luciferase reporter gene detection showed that compared with the miR-27a-3p mimic NC group,transfection with miR-27a-3p mimic down-regulated luciferase activity in wild-type FBXW7-WT plasmid group（P<0.05）,but there was no significant change in luciferase activity in mutant FBXW7-MUT plasmid group（P<0.05）.Compared with h FOB1.19,the expression level of FBXW7 m RNA in osteosarcoma cells MG-63 and Saos-2 was decreased（P<0.05）.Transfection of pc DNA3.1-FLAG-FBXW7 could increase the expression level of FBXW7 in osteosarcoma cells（P<0.05）.The proliferation and migration of osteosarcoma cells were decreased（P<0.05）,and β-catenin protein expression level of osteosarcoma cells were increased（P<0.05）.Transfection of si RNA-FBXW7 decreased the expression of miR-27a-3p in osteosarcoma cells（P<0.05）,increased the proliferation and migration of osteosarcoma cells（P<0.05）,and decreased theβ-catenin protein expression level（P<0.05）.（3）Transfection with miR-27a-3p mimic reduced the expression levels of FBXW7 and β-catenin proteins in osteosarcoma cells.Transfection with miR-27a-3p inhibitor increased the expression levels of FBXW7 and β-catenin protein in osteosarcoma cells（P<0.05）.Transfection with miR-27a-3p inhibitor reduced the proliferation and migration of osteosarcoma cells（P<0.05）,and increased the apoptosis rate of osteosarcoma cells（P<0.05）.The proliferation and migration levels of osteosarcoma cells were partially recovered after miR-27a-3p inhibitor +si FBXW7（P<0.05）.Conclusion（1）miR-27a-3p was highly expressed in osteosarcoma cells,and overexpression of miR-27a-3p promoted proliferation and migration of osteosarcoma cells and inhibited apoptosis,knockdown miR-27a-3p inhibited the proliferation and migration of osteosarcoma cells and promoted apoptosis.（2）FBXW7 is underexpressed in osteosarcoma cells,while overexpression of FBXW7 inhibits proliferation and migration of osteosarcoma cells,and inhibition of FBXW7 expression promotes proliferation and migration of osteosarcoma cells.（3）miR-27a-3p targets the expression of FBXW7 and affects the proliferation,migration and apoptosis of osteosarcoma cells,the mechanism of which may be related to the Wnt/β-catenin signaling pathway. |
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