The Effects Of MiR-708-5p On The Migration And Invasion Of Osteosarcoma Cells And Its Potential Mechanisims

BackgroundsOsteosarcoma?OS?is one of the most common primary malignant bone tumor despite its annual incidence rate is three per million people,but the rate of mortality is extremely high.MicroRNAs?miRNAs?belong to the class of non-coding small RNA molecules of about 22 nt in length,which play critical roles in RNA silencing and the regulation of post-transcription.The expression profile of miRNA has been indentified to be related to diagnosis,staging,progression,prognosis and therapeutic response of osteosarcoma.MiRNA has been recognized as biomarkers in the pathogenesis and progression of osteosarcoma.MiR-708-5p has been reported to play important roles in promoting or inhibiting solid tumors and hematological tumors in recent years.This study aims to investigate the effects of miR-708-5p on the migration and invasion of OS cells and its potential mechanisms.ObjectivesThe expression of miR-708-5p in osteosarcoma cells MG63 and SaOS-2 were measured compared with normal cells HS-5 and hMSC;miR-708-5p was over-expressed in MG63 and SaOS-2 cells with Lipofectamine2000,and the effects and mechanisms of miR-708-5p on the migration and invasion of osteosarcoma cells MG63 and SaOS-2 were examined.Methods1.The expression of miR-708-5p in osteosarcoma cells MG63 and SaOS-2: The miRNAs in dataset GSE70367 from GEO database were analyzed utilizing GEO2 R,with hMSC as the normal control group.The TOP50?log|FC|>1,P<0.05?were chosen from TOP250 analysis,HemI was used to protract the heatmap.qRT-PCR was exploited to measure the expression of miR-708-5p in osteosarcoma cells MG63,SaOS-2 and normal cells HS-5,hMSC.2.The effects of miR-708-5p on the migration and invasion of osteosarcoma cells: miR-708-5p was transfected with Lipofectamine 2000,and RT-qRCR was used to measure the expression levels of miR-708-5p in MG63 and SaOS-2 cells.Wound-healing and Transwell assay were utilized to examine the migration and invasion abilities of MG63,SaOS-2 cells.3.The impacts of miR-708-5p on epithelial-mesenchymal transition?EMT?and matrix metalloprotein?MMP?family in osteosarcoma cell lines MG63 and SaOS-2: RT-qPCR and Western blot were exploited to measure the mRNA and protein levels of MMP2,7,and 9 in MG63 after transfected with miR-708-5p;MMP2 and 9 were measured in SaOS-2 cells similarly.The protein levels of epithelial marker E-cadherin and mesenchymal markers N-cadherin and Vimentin in MG63 and SaOS-2 after transfected with miR-708-5p were detected using Western blot.4.Prediction and validation of the target gene of miR-708-5p: Targetsacan7.2?http://www.targetscan.org/vert₇2/?and dual-luciferase assay were used to predict and validate the target gene of miR-708-5p in osteosarcoma cells.Results1.Gene differential enrichment analysis of miRNAs in dataset GSE70367 revealed that miRNA-708-5p was down-regulated in osteosarcoma cell lines Hu09,SaOS-2,NY,HOS,and MG63,compared to normal cells hMSCs?P<0.05?.MG63 and SaOS-2 cells were selected as our research objects.RT-qPCR results showed that miR-708-5p was down-regulated in osteosarcoma cells MG63 and SaOS-2 compared with normal cells HS-5 and hMSCs?P<0.05?.2.It was indicated from the results of RT-qPCR that the expression of miR-708-5p was significantly over-expressed after transfected with 20 nM of miR-708-5p mimic for 24 h or 48 h in MG63 and SaOS-2 cells?P<0.05?.Compared with the negative control group,the lateral and longitudinal migration abilities of MG63 and SaOS-2 cells significantly decreased after miR-708-5p transfection?P<0.05?.3.Showed from the results of RT-qPCR and Western blot,compared with the negative control group,the mRNA and protein levels of MMP2,7,and 9 decreased after transfection with miR-708-5p in MG63 cells?P<0.05?.Similarly,the mRNA and protein levels of MMP2 and 9 decreased after transfection of miR-708-5p in SaOS-2 cells?P<0.05?.It is revealed from the results of Western blot that the protein level of epithelial marker E-cadherin increased and the levels of mesenchymal markers N-cadherin and Vimentin decreased in MG63 and SaOS-2 cells after miR-708-5p transfected?P<0.05?.4.It was predicted using Targetscan7.2 that miR-708-5p binds to the 3'UTR of SEMA4 C,MAP3K3,and ZEB1 genes.The results of dual-luciferase assay indicated that,miR-708-5p binds directly to 3'UTR of SEMA4 C,MAP3K3,and ZEB1.Western blot showed that the protein levels of SEMA4 C and MAP3K3 were not significantly changed after overexpression of miR-708-5p in MG63 and SaOS-2 cells,while the protein level of ZEB1 was significantly decreased?P < 0.05?.Dual-luciferase reporter assay and Western blot analysis demenstrated that miR-708-5p directly targets ZEB1 in osteosarcoma cells.5.Restoration of ZEB1 expression after overexpression of miR-708-5p in MG63,SaOS-2 cells partially restored the inhibition effects of miR-708-5p on the migration and invasion of MG63 and SaOS-2 cells.6.The migration and invasion abilities of osteosarcoma cells MG63 and SaOS-2 were significantly inhibited after interfering with the expression of ZEB1.Conclusions1.miR-708-5p was lower expressed in osteosarcoma cells MG63 and SaOS-2 compared with normal cells HS-5 and hMSCs.2.miR-708-5p impaired the migration and invasion abilities of MG63 and SaOS-2 cells through inhibiting EMT and MMPs.3.ZEB1 served as one of the directed target gene of miR-708-5p in osteosarcoma cells MG63 and SaOS-2.4.The migration and invasion abilities of MG63 and SaOS-2 cells decreased after transfected with miR-708-5p through directly targeting ZEB1.