The Mechanism Of LncRNA LOC100129620 Regulates Osteosarcoma Metastasis

Objective:Osteosarcoma metastasis is an important cause of death.However,the mechanism of osteosarcoma metastasis is not clear.The lung is one of the main organs of osteosarcoma metastasis.Long chain noncoding RNA(lncRNA)is noncoding RNA with a length of more than 200 nucleotide fragments,which plays an important role in the occurrence and development of the disease.However,the regulatory role of long-chain noncoding RNA in lung metastasis of osteosarcoma is unknown.This study will explore the regulatory role of long-chain noncoding RNA in lung metastasis of osteosarcoma.Methods:The differential expression of lncRNA in osteosarcoma in situ and lung metastasis was analyzed by GEO database to find the long-chain noncoding RNA related to osteosarcoma lung metastasis.The differential expression of lncRNA in osteosarcoma in situ and lung metastasis was detected by real-time fluorescence quantitative PCR.The expression of lncRNA LOC100129620 in osteosarcoma cell line and human bone marrow mesenchymal stem cells was detected by real-time fluorescence quantitative PCR.The localization of LOC100129620 in cells was detected by immunofluorescence in situ hybridization.The expression of LOC100129620 in osteosarcoma cells was regulated by constructing overexpression lentivirus and knockdown lentivirus.CCK-8,clone formation and flow cytometry were used to detect the effects of LOC100129620 on the proliferation and apoptosis of osteosarcoma cells.The effects of LOC100129620 on the migration and invasion of osteosarcoma cells were detected by cell scratch and Transwell.The effect of LOC100129620 osteosarcoma cell proliferation in vivo was detected by constructing a subcutaneous ectopic tumor formation model in nude mice.The binding sites of LOC100129620 and miRNA were analyzed by bioinformatics.The binding of LOC100129620 to miR-335-3p was detected by RNA pull down and double luciferase reporter gene.MiRNA mimics and inhibitors were used to regulate the expression of miR-335-3p in osteosarcoma cells.The effects of miR-335-3p on the proliferation and apoptosis of osteosarcoma cells were detected by CCK-8,clone formation and flow cytometry.The effects of miR-335-3p on the migration and invasion of osteosarcoma cells were detected by cell scratch and Transwell.The binding sites of miR-335-p and CDK6 were analyzed by bioinformatics.The binding of miR-335-p to CDK6 was detected by double luciferase reporter gene.The regulatory effects of LOC100129620 and miR-335-3p on the expression of CDK6 protein were detected by Western blot.HUVEC and macrophage migration were detected by cell scratch and Transwell.The expressions of VEGF and CD206 in tumor tissues were detected by immunofluorescence.The content of M2 macrophages in tumor tissues was detected by flow cytometry.Results:Through GEO analysis,we found that there was a significant difference in the expression of lncRNA between in situ and metastatic lesions of osteosarcoma.The expression of lncRNA LOC100129620 in osteosarcoma lung metastases was significantly higher than that in situ,and the expression of lncRNA LOC100129620 in osteosarcoma cells was significantly higher than that in human bone marrow mesenchymal stem cells.LncRNA LOC100129620 was mainly localized in the cytoplasm of osteosarcoma cells.Promote the expression of lncrna loc100129620,enhance the proliferation,migration and invasion of osteosarcoma cells,inhibit the expression of lncRNA LOC100129620,and reduce the proliferation,migration and invasion of osteosarcoma cells.LOC100129620 has potential binding sites with multiple miRNAs,and has the strongest binding ability with miR-335-3p.MiR-335-3p overexpression reduce the proliferation,migration and invasion of osteosarcoma cells,inhibited the expression of miR-335-3p,and enhance the proliferation,migration and invasion of osteosarcoma cells.Mir-335-3p binds to the 3’-UTR region of CDK6 mRNA.Overexpression of miR-335-3p reduces the expression of Cdk6 protein,and knockdown of miR-335-3p promotes the expression of CDK6 protein.Overexpression of LOC100129620 promoted the expression of CDK6 protein,and knockdown of LOC100129620 decreased the expression of CDK6 protein.Overexpression of LOC100129620 enhanced the effect of osteosarcoma cells on the migration ability of HUVEC and macrophages,and knockdown of LOC100129620 decreased the effect of osteosarcoma cells on the migration ability of HUVEC and macrophages.Overexpression of LOC100129620 enhanced the expression of VEGF and CD206 in bone tumors,and knockdown of LOC100129620 decreased the expression of VEGF and cd206 in bone tumors.Conclusion:lncRNA loc100129620 regulates miR-335-3p/CDK6 signaling pathway through ceRNA mechanism and mediates the proliferation,migration and invasion of osteosarcoma cells.LncRNA LOC100129620 promotes lung metastasis of osteosarcoma by promoting angiogenesis and macrophage migration in tumor tissue.