MicroRNA-208b Inhibits Proliferation,Migration And Invasion Of Osteosarcoma Cells By Negatively Regulating Of ROR2 In Human

Objective Osteosarcoma is a kind of primary bone tumor with high malignancy in children and adolescents.The tumor is prone to distant lung metastasis in the early stage,with high mortality and poor prognosis.It is an important way to improve the prognosis of osteosarcoma by exploring the specific pathogenesis and new treatment strategies.MicroRNA(miRNA)is a kind of endogenous non coding small RNA,which is combined with the 3' untranslated region(3' UTR)of the target gene mRNA,and which can caused the degradation of target gene mRNA or translation blocked,playing a negative regulatory role.It has been reported that the dysregulation of miRNA plays an important role in the occurrence,development,invasion and metastasis of osteosarcoma.The purpose of this study was to investigate the expression of miR-208b in osteosarcoma tissues and cell lines and its effects on proliferation,invasion and metastasis of osteosarcoma cells.Methods Firstly,we detected the differential expression of miR-208b in osteosarcoma tissues,normal bone tissues,normal human osteoblast cell line NHOST and two osteosarcoma cell lines(U20S and Saos-2)by using quantitative real-time PCR(qRT-PCR).Subsequently,we detected the effect of over-expressed miR-208b on the proliferation of osteosarcoma U20S and Saos-2 cells by CCK8 assay and clone formation assay;and the cell cycle of osteosarcoma cells was detected through flow cytometry;and the subcutaneously implanted model of Saos-2 was used to observe the tumor forming ability in nude mice in vivo;and wound healing assay and Transwell invasion assay were used to detect the invasion and migration abilities of osteosarcoma cells.Furthermore,we predicted the target genes of miR-208b by bioinformatics analysis,and detected the binding activity of miR-208b to ROR2 mRNA 3' UTR through luciferase reporter assay.Through quantative real-time PCR and western blotting,we detected the ROR2 mRNA and protein level with overexpression of miR-208b.Then,using small RNA interference(siRNA)technology to design and synthesis of ROR2-siRNA,and the ROR2-siRNA or siRNA control was transfected into U2OS and Saos-2 cells;and we detected the effects of ROR2 gene silence on proliferation,apoptosis,invasion and metastasis of osteosarcoma cells.Finally,recovery experiments in osteosarcoma cells were transfected with mimics control,miR-208b mimics,miR-208b mimics+pcDNA3.0 and miR-208b mimics+pcDNA3.0/ROR2,using a series of tests to detect changes of osteosarcoma cell proliferation,migration and invasion.Results1.Overexpression of miR-208b inhibited the growth and proliferation of osteosarcoma cells in vitro and vivo:In this study,we found that the relative level of miR-208b in osteosarcoma tissues was significantly lower than that in normal bone tissues;and the relative levels of miR-208b in osteosarcoma cell lines U2OS and Saos-2 were significantly lower than those of normal human osteoblast cell line NHOst(both P<0.05).In osteosarcoma cell lines U20S and Saos-2,compared with the control group,the growth activity and the clone formation rate of the cells were significantly lower.We also built the subcutaneously implanted models of Saos-2 cells.The tumors in miR-208b mimics group had smaller size and lower weight than control group.2.Overexpression of miR-208b inhibited cell cycle progression in osteosarcoma cells:The flow cytometry results showed that in U20S and Saos-2 cells,compared with control group,the proportion of G0/G1 phase increased significantly after transfection with miR-208b mimics,while the proportion of S phase significantly decreased.3.Overexpression of miR-208b inhibited the migration and invasion of osteosarcoma cells:The results showed that compared with mimics control group,the wound healing rate and invasion ability of U20S and Saos-2 cells transfected with miR-208b mimics were both obviously decreased.4.MiR-208b can bind to the 3' UTR of ROR2 mRNA:In the database of PicTar and miRanda,we found miR-208b can bind to ROR2 mRNA 3' UTR.The results of luciferase assay showed that miR-208b can target ROR2 mRNA 3' UTR and inhibit the activity of luciferase(P<0.05).But,there was no statistically significant difference in mutant vectors.5.MiR-208b negatively regulated ROR2 expression at post transcriptional level:Compared with the control group,ROR2 protein level was significantly decreased after transfection with miR-208b mimics in U20S and Saos-2 cells.6.Knockdown of ROR2 inhibited proliferation of osteosarcoma cells:In U20S and Saos-2 cells,we used siRNA to knockdown the ROR2 expression.The relative level of ROR2 protein significantly decreased.The results showed that knockdown of ROR2 inhibited growth activity of osteosarcoma cells.7.Knockdown of ROR2 inhibited migration and invasion of osteosarcoma cells:The results showed that compared with mimics control group,the wound healing rate and invasion ability of U20S and Saos-2 cells transfected with ROR2-siRNA were both obviously decreased(P<0.05).8.MiR-208b regulated proliferation,migration and invasion of osteosarcoma cells by targeting ROR2:Co-transfection of miR-208b mimics and pcDNA3.0/ROR2,the expression of ROR2 protein was rescued.The results showed that the growth activity,the clone formation rate,wound healing rate and invasion ability of the osteosarcoma cells were rescued.MiR-208b regulated cell proliferation,invasion and migration by targeting ROR2.Conclusions1.The relative level of miR-208b was significantly low expression in osteosarcoma tissues and two osteosarcoma cell lines(U20S and Saos-2),and overexpression of miR-208b can significantly inhibit proliferation,invasion and migration of osteosarcoma cells;and inhibit osteosarcoma xenografts in nude mice.2.Bioinformatics analysis predicted that the ROR2 was a direct target gene of miR-208b,and was negatively regulated by miR-208b at the post transcriptional level.3.After the expression of ROR2-siRNA alone,the proliferation,invasion and migration of osteosarcoma cells were significantly inhibited.These results were consistent with over expression of miR-208b.4.On the basis of over expression of miR-208b,the expression level of ROR2 protein was restored,and the proliferation,invasion and migration of osteosarcoma cells were significantly restored.In conclusion,miR-208b can inhibit the proliferation,invasion and migration of osteosarcoma cells by negatively regulating of ROR2.