Up-regulated ONZIN Promotes Proliferation Of Osteosarcoma Cell By Regulating CDK/Rb/E2F Pathway

Objective : Osteosarcoma is a common malignant bone tumor with poor prognosis.Osteosarcoma almost occurs in adolescents and children.Osteosarcoma is prone to metastases to lung,resulting in extremely high mortality.The treatment and outcomes of osteosarcoma have not changed substantially over the past 30 years.ONZIN have been demonstrated to be relevant with the occurrence and development of osteosarcoma,however,its mechanism is unclear yet.We sought to elucidate the role of ONZIN involved in osteosarcoma development.The purpose of this study was to investigate potential signaling pathways and related genes that ONZIN act on in the development of osteosarcoma.Methods: In this study,we successfully established osteosarcoma cell line MG-63 with stably ONZIN overexpression by transfecting specific plasmid.Quantitive Real-Time PCR(qPCR)and Western blot were used to verify the effect of infection.RNAs was extracted from ONZIN overexpression cells and its control.Next these samples were detected using Nanodrop2000 and agarose gel electrophoresis.RNA sequencing was then performed to further search the mechanism by which ONZIN regulates the development of osteosarcoma.According to the results of quality controls and evaluation by FastQC,we analyzed clean data to find the differentially expression genes(DEGs)by RSEM.Bioinformatics tools including Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were used to identify the functions and signaling pathways which were conducted by ONZIN in osteosarcoma.QPCR and western blot was performed to analysis the expression of CDK/Rb/E2F signaling pathway components in MG-63 cells transfected with vector or ONZIN.The phosphorylation status of Rb at ser780 and ser811 was detected using Western blot.In addition,cell proliferation of MG-63 cells transfected with vector or ONZIN was detected by CCK8.Results: After raw data were filtered by Fast QC,we obtained a total of 44343754 clean reads,accounting for 98.% of the total reads.The quality of RNA-sequencing meet the requirements for further analysis.Totally 2925 differentially expressed genes(DEGs)were screened between ONZIN-overexpression cells MG-63 and its controls,among them 1314 were up-regulated and 1611 were down-regulated.GO enrichment analysis showed that: DEGs were significantly enriched in cell,cell part and organelle from the aspect of cell components;in cellular process,single-organism process and metabolic process from the aspect of cell components,in binding and catalytic activities from the aspect of molecular functions.KEGG Pathway analysis showed that the pathways in cancer was significantly enriched,and 151 genes were enriched to this pathway,accounting for 5.83%.Among pathways in cancer,the CDK/Rb/E2F signaling pathway was deeply affected involving in cell proliferation.Furthermore,the results of western blot and qPCR showed that the expression of CDK/Rb/E2F signaling pathway components is increased by overexpressing ONZIN in osteosarcoma.And then,we found that onzin increased CDK-mediated Rb phosphorylation by detecting the phosphorylation status of Rb at ser780 and ser811.The results of CCK8 showed that overexpressed ONZIN promotes cell proliferation in osteosarcoma cell line MG-63.Conclusion: This study demonstrate that CDK/Rb/E2F pathway was activated by ONZIN which promotes cell proliferation in the progression of osteosarcoma.The study expounded a feasible pattern among complicated regulation networks and helps to understand the role of ONZIN involved in osteosarcoma development.Our findings suggest that ONZIN and CDK/Rb/E2F pathway might be a potential therapeutic targets for the treatment of osteosarcoma patients.More regulatory mechanisms of ONZN in osteosarcoma ought to be clarified to provide the means for novel therapeutic approaches in fight with the malignant disease.