Decreased Expression Of MiR-195 Mediated By Hypermethylating Promote Malignant Phenotype And Molecular Mechanisms In Osteosarcoma Cell

BackgroundOsteosarcoma(OS)is the most common type of primary malignant bone tumor in children and adolescents,whose Earily lung metastasis is its main factors of poor prognosis.Therefore,searching for new target and new mechanism of OS metastasis is essential for OS prevention and treatment.Reports found that the expression level of miR-195 was negatively correlated with OS metastasis.Up-regulating miR-195 could inhibit the apoptosis,migration and invasion of OS cells.Fatty acid synthase(FASN)was highly expressed in osteosarcoma and its expression level was positively correlated with lung metastasis.Silencing FASN could inhibit the proliferation,migration and invasion of osteosarcoma cells in vitro.Aberrant DNA methylation can reverse regulate the expression of miRNAs,which plays an important role in the pathogenesis and metastasis of osteosarcoma.However,if miR-195 could targeting regulate FASN expression and ectopic DNA methylation is the upstream regulatory mechanism of miR-195 in osteosarcoma metastasis is currently reported less.Objective:1.To detect the expression levels of miR-195 and FASN in fresh osteosarcoma tissue and normal bone tissue and the methylation level of miR-195 DNA promoter region.2.To investigate the effect of miR-195 molecular on migration and invasion of OS cells in vitro by targeting on FASN.3.To investigate the methylation of miR-195 promoter region is one of the mechanisms that affect the invasion and migration of osteosarcoma cells in vitro.Materials and methods:1.The expression of miR-195 in fresh osteosarcoma and its methylation level.The expression of miR-195 and FASN were detected by RT-PCR and western blot.The methylation level of miR-195 DNA promoter was detected by MSP method.2.The Effects of miR-195 molecular on migration and invasion of OS cells in vitro by targeting on FASN.(1)The Bioinformatics was used to predict the possible combined site between miR-195 and FASN,and then luciferase activity assay was used to further confirm the targeted regulation between the two.(2)The overexpression and inhibition vectors containing miR-195 were constructed,and transfected to U2-OS/143B,MTT,Transwell invasion and wound healing assay were used to detect the proliferation,migration and invasion of OS cells.3.Methylation regulation of miR-195 is one of the mechanisms to impact invasion and migration of osteosarcoma cells in vitro.(1)The online prediction of CPG island was used to predict possibility of methylation of miR-195 promoter region.Then methylation inhibitor 5-aza-2'-dc was used to treat human osteosarcoma cells.BSP was used to detect methylation level,The expression of miR-195 was detected by PCR.(2)The proliferation,migration and invasion of human osteosarcoma cells treated by methylation inhibitor 5-aza-2'-dc were detected by MTT,Transwell invasion and wound healing assays.Statistical analysis:All analyses were performed with SPSS Version 19.0(SPSS Inc,Chicago,IL,USA).Data are expressed as the mean ± SD.One-way ANOVA or the student's T test were used in multiple-sample analysis.A value of P<0.05 was considered as significant difference.Result:1.The expression of miR-195 in fresh osteosarcoma tissue was significantly lower than that in fresh normal bone tissue,while the expression level of FASN was significantly increased in the fresh osteosarcoma tissue.The methylation level of miR-195 promoter region in fresh osteosarcoma tissue was significantly higher than that in fresh normal Bone tissue.2.The effects of miR-195 on migration and invasion of osteosarcoma cells in vitro by targeting on FASN gene.(1)miRNA-195 has a conserved binding site between FASN 3'UTR 153bp-159bp;miR-195 could negatively regulate FASN expression.(2)The proliferation,invasion and migration ability of osteosarcoma cells were significantly decreased when up-expressing miR-195,while these were significantly improved after miR-195 was down-regulated.3.Methylation regulation of miR-195 is one of the mechanisms to impact invasion and migration of osteosarcoma cells in vitro.(1)The miR-195 molecular promoter region is rich in CPG islands;The methylation level of the promoter region of miR-195 in OS cell was significantly decreased when treated by methylation inhibitor 5-aza-2'-dc,and the expression level of miR-195 in OS cells was significantly up-regulated,meanwhile.(2)The proliferation,migration and invasion ability of osteosarcoma cells were significantly decreased after treating with methylation inhibitor 5-aza-2'-dc.Conclusion1.MiR-195 can inhibit the proliferation,migration and invasion ability of osteosarcoma cells by negative regulating of FASN gene.2.The hypermethylation epigenetic regulation of miR-195 promoter region is one of the mechanisms of its low expression in osteosarcoma cells,and inhibition of miR-195 methylation can inhibit the proliferation,migration and invasion ability of osteosarcoma cells.