| Objective:We planned to integrate the existing database of osteosarcoma metastasis,explore the expression changes of long noncoding RNAs(lncRNAs),and clarify how they are involved in the occurrence and development of osteosarcoma.Then we focused on the candidates and carried out the study of their functions and mechanisms,which would help to expand the regulatory network of osteosarcoma development,deepen the understanding of this disease,and provide new potential therapeutic targets for its treatment.Contents:We found that the expression of long non-coding RNA LINC00607 was significantly up-regulated in lung metastasis of osteosarcoma,and the subcellular localization of LINC00607 was confirmed by expression profile analysis.Functional experiments confirmed the effects on the behaviors of tumor or endothelial cells,and epithelial-mesenchymal transition(EMT).In terms of mechanism study,the downstream regulatory genes of LINC00607 and how to regulate the expression of the downstream gene were explored.The effect of LINC00607 on bone tumor development was further clarified by in vivo experiments.Methods:The expression of lncRNA in osteosarcoma was verified by real-time fluorescent quantitative PCR.The subcellular localization of LINC00607 was confirmed by nuclear cytoplasmic separation and quantification combined with fluorescence in situ hybridization.Lentivirus overexpression and knockdown of lncrna LINC00607 in osteosarcoma cell lines were used.The effects of LINC00607 on proliferation,migration and invasion of osteosarcoma cells were detected by CCK-8 assay,Transwell assay,flow cytometry,cell scratch assay and colony formation assay.The effects of LINC00607 on EMT transition related gene expression in osteosarcoma cells were detected by real-time PCR and Western blot.The miRNA binding to LINC00607 was analyzed by bioinformatics,and the binding of LINC00607 to miRNA was detected by RNA pull down and dual luciferase reporter gene.We used miRNA mimics and inhibitors to regulate the expression of miRNA in osteosarcoma cells,and detected the effects of miRNA on the proliferation,migration and invasion of osteosarcoma cells.Bioinformatics was used to analyze the target genes of miRNA,and dual luciferase reporter gene was used to detect the binding of miRNA and target genes.Results:Through the analysis of RNA sequencing data,we found that the expression of lncrna in osteosarcoma metastasis and in situ has significant difference.The expression of lncrna was significantly increased in osteosarcoma tissues.Real time PCR and fluorescence in situ hybridization showed that LINC00607 was mainly located in the cytoplasm.We found that overexpression of LINC00607 can promote the proliferation,migration and invasion of osteosarcoma cells,while knockdown of LINC00607 expression in osteosarcoma cells can inhibit the proliferation,migration and invasion of osteosarcoma cells.Overexpression of LINC00607 aggravates epithelial mesenchymal transition,while inhibition of LINC00607 reduces epithelial mesenchymal transition.Mechanism studies show that LINC00607 can adsorb miR-607 and regulate its function.Overexpression of miR-607 can inhibit the proliferation,migration and invasion of osteosarcoma cells,while knockdown of miR-607 expression in osteosarcoma cells can promote the proliferation,migration and invasion of osteosarcoma cells.Bioinformatics analysis and experiments show that miR-607 directly targets the 3’UTR region of transcription factor E2F6 and regulates the expression of E2F6.Further rescue experiments show that E2F6 plays a role in the downstream of LINC00607/miR-607 and participates in the regulation of proliferation and invasion of osteosarcoma cells.Conclusions:This study reveals the important role of long non-coding RNA in the development of osteosarcoma and demonstrates that LINC00607 located in the cytoplasm sponges miR-607 and upregulates E2F6 expression,thereby promoting tumor proliferation and invasion in osteosarcoma.In this study,the regulatory factors of osteosarcoma were extended to long non-coding RNA,which provided a theoretical basis for the research on the occurrence and development of osteosarcoma and a new view for its treatment. |
