Overexpression Of MiR-506 Suppresses Proliferation And Promotes Apoptosis Of Osteosarcoma By Targeting Astrocyte Elevated Gene-1(AEG-1)

Osteosarcoma is the most common primary malignancy of the bone in children and adolescents,characterized by a highly malignant tendency to destroy the surrounding tissues and to metastasize almost exclusively to the lung,which is the main cause of death.In young patients,it is most often localized in the distal femur and proximal tibia region.Although osteosarcoma has been treated with neoadjuvant chemotherapy in combination with surgery for more than 3 decades,patients with recurrent or metastatic osteosarcoma still have an extremely poor prognosis,with a long-term survival of less than 10%.To date,the molecular mechanisms underlying the initiation,development and metastasis of osteosarcoma are not completely known.Therefore,it is essential to identify new therapeutic targets and develop therapeutic strategies against osteosarcoma.micro RNAs(miRNAs)belong to a group of small noncoding,single-stranded RNA fragments measuring 18 to 25 nucleotides that play critical regulatory roles in tumorigenesis and cancer progression.Recently,these miRNAs have been shown to suppress translation or directly cleave the target m RNA through complementary sequence pairing to the 3'-UTR or coding region of m RNA targets.These data indicate that miRNAs can be used as diagnostic biomarkers and may function either as oncogenes or as tumor suppressors based on the effects of their target m RNAs.It is reported that in osteosarcoma,multiple miRNAs,such as miR-29,miR-125 b,miR-143,miR-199a-3p and so on,are involved in tumor growth,progression and metastasis.A previous study revealed that restoration of miR-506 in malignant transformed human bronchial epithelial cells suppressed cell proliferation.Overexpression of miR-506 inhibited TGF-?-induced epithelial-mesenchymal transition and suppressed adhesion,invasion and migration of human breast cancer cells.Moreover,overexpression of miR-506 in established hydroxycamptothecinresistant colon cancer cells confered resistance to hydroxycamptothecin through inhibiting the expression of peroxisome proliferator-activated receptor ?(PPAR?).Although miR-506 has been subjected to extensively study in recent years,its role in the initiation and progression of osteosarcoma,and the molecular mechanisms by which miR-506 exerts its effects,are poorly understood.The research includes three parts.The first part is the function research of AEG-1 and miR-506 in osteosarcoma.The second part is that miR-506 plays an significant role in osteosarcoma by targeting AEG-1.The last part is miR-506?AEG-1 inhibits osteosarcoma by regulating the Wnt/?-catenin signaling pathway.Part One The function research of AEG-1 and miR-506 in osteosarcoma.Methods: 1.The expression level of AEG-1 and miR-506 in osteosarcoma tissues and osteosarcoma cell line MG63 was measured by using real-time PCR and western blot analysis.2.MTT assay was used to detect the effects of down-regulation of AEG-1 or miR-506 overexpression on the viability of osteosarcoma cell line MG63 cells.3.Flow cytometry was used to measure the effects of down-regulation of AEG-1 or miR-506 overexpression on osteosarcoma cell line MG63 cell apoptosis.4.To explore the role of miR-506 in osteosarcoma in vivo,a mouse model of osteosarcoma was constructed.Results: 1.The level of miR-506 is inversely correlated with AEG-1 protein expression in osteosarcoma.2.AEG-1 knockdown or up-regulation of miR-506 suppresses proliferation of osteosarcoma cells.3.AEG-1 knockdown or up-regulation of miR-506 inhibits apoptosis of osteosarcoma cells.4.miR-506 inhibits osteosarcoma cell growth in vivo.Part Two miR-506 plays a significant role in osteosarcoma by targeting AEG-1.Methods: 1.Using bioinformatics analysis,the target genes of miR-506 were predicted.2.Dual luciferase report experiments were used to identify the target genes of miR-506.3.Western blot and real-time PCR were used to detect the expression level of AEG-1 after overexpression and down-regulation of miR-506.Results: 1.Bioinformatics analysis by using Target Scan suggested that miR-506 was predicted to target the AEG-1.2.Luciferase reporter assays confirmed that AEG-1 was a direct downstream target of miR-506.3.Overexpression of miR-506 suppressed the expression of AEG-1 in MG63 cells.Part Three miR-506?AEG-1 inhibits osteosarcoma by regulating the Wnt/?-catenin signaling pathway.Methods: 1.Western blot and real-time PCR were used to detect the effect of up-regulation of miR-506 and down-regulation of AEG-1 on expression levels of ?-catenin,c-myc,and cyclin D1 in MG63 cells.2.Flow cytometry was used to measure the effects of the Wnt/?-catenin signaling pathway inhibition by si-?-catenin on osteosarcoma cell line MG63 cell apoptosis.3.Flow cytometry was used to measure the effects of CGP049090,a small molecule inhibitor of Wnt/?-catenin,on the fuctions of osteosarcoma cell line MG63.Results: 1.Both up-regulation of miR-506 and down-regulation of AEG-1 clearly decreased the expression levels of ?-catenin,c-myc,and cyclin D1 in MG63 cells.2.Blocking of the Wnt/?-catenin signaling pathway by si-?-catenin suppressed proliferation and induced apoptosis of MG63 cells.3.CGP049090,a small molecule inhibitor of Wnt/?-catenin,obviously inhibited proliferation and induced apoptosis of MG63 cells.Conclusion 1.The level of miR-506 is inversely correlated with AEG-1 protein expression in osteosarcoma.2.Up-regulation of miR-506 suppresses proliferation of osteosarcoma cells.3.Up-regulation of miR-506 inhibits apoptosis of osteosarcoma cells.4.AEG-1 is directly targeted by miR-506 in MG63 cells.5.miR-506 inhibits osteosarcoma cell growth in vivo.6.miR-506 inhibits osteosarcoma by regulating the Wnt/?-catenin signaling pathway.