The Study Of MiR-183Expression And Effects On Metastasis In Osteosarcoma Cells In Vitro

Background and Objective:Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. The estimated incidence rate worldwide is4/million/year, with a peak incidence at the age of15-19years. In OS there is a high tendency to metastatic spread. Approximately20%of patients present with lung metastases at initial diagnosis and, additionally, in40%of patients metastases occur at a later stage. Eighty percent of all metastases arise in the lungs, most commonly in the periphery of the lungs, and exhibit resistance to conventional chemotherapy. The5-year survival rate for OS patients with metastases is20%compared to65%for patients with localised disease and most deaths associated with OS are the result of metastatic disease.Metastasis is considered to be the final though most critical step in tumorigenesis of malignant tumours. It has been shown that OS lung metastases display a biological behaviour different from the primary tumours. Metastases are comprised of cell clones that differ from primary tumours with respect to ploidy, enzyme profile, karyotype and chemosensitivity. Therapeutic regimens that target primary tumours are therefore unlikely to be successful in the treatment of metastatic disease.The metastatic cancer cells subsequently complete many steps to metastasis. Over the last decade, much research has been performed to try to unravel the biology of OS metastasis and many (pre)clinical studies have attempted to discover new treatment options for metastatic OS. However, the exact metastatic mechanism of OS remains unclear. Further study about the molecular mechanism of OS will provide novel treatments for therapy in metastatic OS.miRNA expression profiling analyses have reported a general dysregulation of miRNA expression in all tumors. In this study we will outline the biological and molecular roles of miRNAs in OS and metastasis by highlighting the most promising advances for the use of miRNAs in the clinical setting. The aim of this study is to explore the biology of metastatic OS cells and how these may contribute to designing a metastasis directed treatment for OS.Methods:Firstly, we compared the differences of miR-183and Ezrin expression in high-metastatic human OS F5M2and low-metastatic human OS F4cells. Functional analysis was then carried out by transfection of miR-183mimics or inhibitors into the high-metastasis OS F5M2cell line with low endogenous miR-183expression. The results of the transfection were subsequently assessed on cell viability pattern by proliferation and apoptosis assay, cell migration by transwell insert and wound healing assay, gene expression alterations by real-time PCR and protein levels by western blotting and immunocytochemistry (ICC). At last we study the relationship between the miR-183level and the metastatic potential of OS.Results:1. The expression level of Ezrin in high metastatic OS was significantly higher than that of in low metastatic OS. There is a same tendency either in cells or in samples. Real-time PCR demonstrated that miR-183expression in high metastatic F5M2cells was significantly lower than in low metastatic F4cells.2. It showed that miR-183was significantly over-expressed in F5M2cells after transfection with the miR-183mimics. In this study, we successfully established OS cell models with different expression of miR-183. 3. Transwell insert and wound healing assay demonstrated that ectopic expression of miR-183inhibited F5M2cell migration and invasion potential in vitro. The results of flow cytometry and MTT assay showed that there was no difference on apoptosis and proliferation between F5M2cells untreated or transfected with miR-183mimics.4. Western blotting and ICC showed that the expression of Ezrin in F5M2cells decreased markedly after transfection with miR-183mimics, compared with cells untreated or treated with NC. There was an inverse correlation between Ezrin production and miR-183levels.Conclusion:Overexpression of miR-183mainly inhibited the migration and invasion of F5M2cells, not proliferation and apoptosis. Ezrin expression was positively correlated to migration and invasion of OS cells. It is concluded that alteration of miR-183might act as a significant inhibitory factor in regulate cell migration and invasion targeting the downregulation of Ezrin expression. To our knowledge, this is the first in vitro study to regulate metastasis and progression of OS, by upregulation of miR-183to target the expression of Ezrin in F5M2cells. Our study might provide an important avenue for further analysis in vivo with the aim to develop a new potential diagnostic and therapeutic targets for the screening and treatment of high metastatic OS. Further studies are required to fully understand the regulation mechanisms of miR-183and Ezrin in OS in vitro and in vivo.