Biological Function And Clinical Significance Of CircRNA Hsa_circ₀001017 In Osteosarcoma

BackgroundOsteosarcoma occurs in adolescents with high disability and mortality,and poor prognosis.With the development of sequencing technology and molecular biotechnology,circular RNA has recently become a research hotspot in tumorigenesis and development,as a potential tumor marker,it is of great clinical significance to find circular RNAs that can be used for diagnosis and treatment targets of osteosarcoma.PurposeTo analyze circular RNA differential expression profiles in osteosarcoma tissue samples by high-throughput sequencing technology.To study the biological function of hsa_circ₀001017 in osteosarcoma cells.To explore the clinical significance of hsa_circ₀001017 by analyzing its expression changes in osteosarcoma tissues and peripheral serum.MethodThe second-generation high-throughput sequencing technology was used to detect the circular RNA expression profiles of 3 cases of osteosarcoma tissue and 1 case of normal bone tissue samples;5 circular RNAs with significant differences and good consistency between samples were selected.The qRT-PCR was performed in 3 osteosarcoma cell lines and 6 osteosarcoma patient tissue samples to select circular RNAs that could be used as tumor markers.2)The hsa_circ₀001017 overexpression and interference vector was constructed and used to transfect human osteosarcoma U2OS cells.MTT assay was used to detect cell proliferation after transfection,Transwell assay was used to detect migration and invasion,and flow cytometry was used to detect apoptosis.3)39 patients with osteosarcoma and 24 healthy volunteers were recruited.The qRT-PCR was used to detect the expression of hsa_circ₀001017 in tumor tissues,tumor tissues,and peripheral plasma.The ROC curve was used to analyze the diagnostic value of hsa_circ₀001017 for osteosarcoma.Multivariate regression analysis was used to study the correlation between hsa_circ₀001017 and tumor pathological parameters,and the survival curve and COX regression analysis were used to analyze the effect of hsa_circ₀001017 on the survival rate of patients with osteosarcoma.ResultA total of 246 circular RNAs were differentially expressed between 3 osteosarcoma tissue samples and normal bone tissue samples,of which 80 were up-regulated and 166 were down-regulated.In 6 osteosarcoma tissues and 3 osteosarcoma cell lines,hsa_circ₀000704,hsa_circ₀001017,and hsa_circ₀005035 were all up-regulated,and hsa_circ₀001016 and hsa_circ₀008934 were down-regulated.These were consistent with the sequencing results.2)The hsa_circ₀001017 overexpression vector and interference vector were successfully constructed and transfected into human osteosarcoma U2OS cells.MTT experiments showed that the proliferation ability of U2OS cells was significantly increased after hsa_circ₀001017 overexpression,while the proliferation ability was decreased after hsa_circ₀001017 knockdown.Tanswell experiments showed that the cell migration and invasion ability increased significantly after hsa_circ₀001017 overexpression,while the migration and invasion ability decreased after hsa_circ₀001017 knockdown.Flow cytometry showed that apoptosis was reduced after hsa_circ₀001017 overexpression,while apoptosis was increased after hsa circ 0001017 knockdown.3)Compared with healthy volunteers,the expression of hsa_circ₀001017 in peripheral serum of patients with osteosarcoma was significantly up-regulated.Compared with adjacent tumor tissue,the expression of hsa_circ₀001017 in tumor tissue and peripheral plasma of patients with osteosarcoma was significantly up-regulated and linearly correlated.The ROC curve was drawn and found that the AUC of hsa_circ₀001017 in peripheral plasma was 0.8579,and the AUC of hsa_circ₀001017 in tumor tissue was 0.8097.Osteosarcoma patients were divided into high expression group and low expression group according to the plasma hsa_circ₀001017 expression level.Logistic multivariate regression analysis showed that the upregulation of hsa_circ₀001017 expression was positively correlated with osteosarcoma Price classification and lung metastasis;survival analysis showed The 5-year overall survival rate of the hsa_circ₀001017 high expression group decreased significantly,and COX regression analysis suggested that up-regulation of plasma hsa_circ₀001017 expression was an independent risk factor affecting survival in osteosarcoma patients.ConclusionThe differential expression profile of circular RNA of osteosarcoma constructed by high-throughput sequencing was reliable.2)Circular RNA hsa_circ₀001017 was up-regulated in osteosarcoma tissue samples and osteosarcoma cell lines,which was consistent with the sequencing results.3)Cell experiments in vitro indicated that hsa_circ₀001017 overexpression could promote the proliferation,migration,and invasion of osteosarcoma U2OS cells,but inhibited cell apoptosis,while hsa_circ₀001017 knockdown had the opposite effect,suggesting that hsa_circ₀001017 acted as an oncogene in osteosarcoma.4)Clinically,the expression of hsa_circ₀001017 in peripheral plasma and tissue samples of osteosarcoma patients was consistently up-regulated,and the up-regulation of hsa_circ₀001017 had high diagnostic value for osteosarcoma.And the more obvious the up-regulation of hsa_circ₀001017,the more serious the osteosarcoma disease,and the worse the prognosis.