MiR-34a-5p Targets FoxM1 To Regulate The Proliferation,Invasion,Migration And Apoptosis Of Osteosarcoma Cells

Objective: Osteosarcoma(OS),which occurs in children and adolescents,is the most common primary malignant bone tumor and has a high disability and mortality rate.Micro RNA(miRNA)is an important non-coding RNA(nc RNA)that can influence the progression of many tumors by regulating the expression of various transcription factors.The miR-34a-5p was found to be closely associated with osteosarcoma,but its specific mechanism remains unclear.Therefore,in this paper,we investigated the specific regulatory effects of miR-34a-5p on the proliferation,invasion,migration and apoptosis of osteosarcoma cells and the related mechanisms through in vitro cellular assays.Methods:(1)The expression differences of miR-34a-5p and FoxM1 in nine collected human osteosarcoma tissues and paraneoplastic tissues were compared by quantitative real-time fluorescence polymerase chain reaction(q RT-PCR),respectively.(2)Transient transfection was used to up-and down-regulate miR-34a-5p in human osteosarcoma cell lines MG-63 and U2 OS.The transfection efficiency was verified by q RT-PCR,and the effects of different transfection conditions on the expression of miR-34a-5p and FoxM1 were investigated by CCK-8(Cell Counting Kit-8)assay,cell scratch assay,Transwell assay and flow cytometry,respectively,in MG-63 and U2 OS cells.-63 and U2 OS cells on proliferation,invasion,migration and apoptosis.Changes in the expression levels of relevant phenotypic proteins were analyzed using protein immunoblotting assays(Western blotting,Wb)to verify.(3)The downstream target genes of miR-34a-5p were predicted by bioinformatics techniques,and the targeting relationship was verified by dual luciferase reporter gene assay,q RT-PCR and protein immunoblotting assay.(4)To set up a "rescue assay" to further illustrate the regulatory role of FoxM1 in osteosarcoma and the targeting relationship between miR-34a-5p and FoxM1 by CCK-8 assay,cell scratch assay,Transwell assay and flow cytometry,as well as q RT-PCR and protein immunoblotting assays.relationship.Results:(1)qRT-PCR results showed that miR-34a-5p expression was significantly down-regulated and FoxM1 expression was up-regulated in osteosarcoma tissues and normal paracancerous tissues in 9 cases.(2)The q RT-PCR results showed good transfection efficiency in all groups of cells.Compared with the nonsense transfection group,overexpression of miR-34a-5p inhibited the proliferation,migration and invasion ability of cells in both MG-63 and U2 OS cells and exerted a pro-apoptotic effect.Inhibition of miR-34a-5p,on the other hand,significantly promoted the proliferation,migration and invasion ability of MG-63 and U2 OS cells,exerting an anti-apoptotic effect.(3)Bioinformatic analysis showed that FoxM1 might be a downstream target gene of miR-34a-5p.The dual-luciferase reporter gene assay demonstrated that miR-34a-5p could bind to FoxM1.q RT-PCR and protein immunoblotting experiments confirmed that miR-34a-5p negatively regulated the expression of FoxM1.(4)The "rescue assay" demonstrated that knockdown of FoxM1 significantly inhibited the proliferation,migration and invasion of MG-63 and U2 OS cells and exerted pro-apoptotic effects,while co-inhibition of miR-34a-5p and FoxM1 partially reversed the inhibitory effects of knockdown of FoxM1 alone on MG-63 and U2 OS cells.miR-34a-5p regulated the expression levels of proliferation-associated protein PCNA,migration-associated protein MMP9,and pro-apoptotic protein Caspase-3 in osteosarcoma cells by targeting FoxM1.Conclusions:(1)miR-34a-5p expression was down-regulated and FoxM1 expression was up-regulated in osteosarcoma tissues.(2)miR-34a-5p may play an oncogenic role in osteosarcoma.(3)FoxM1 is a direct target of miR-34a-5p,and miR-34a-5p negatively regulates the expression of FoxM1.(4)Mi R-34a-5p affects the expression of PCNA,MMP9 and Caspase-3 by targeting FoxM1,and thus regulates the proliferation,invasion,migration and apoptosis of osteosarcoma cells.