Meta Analysis Of Circular RNA In The Diagnosis And Prognosis Of Osteosarcoma And The Effect Of Hsa＿circ＿0032462 On Proliferation,Migration And Apoptosis Of Osteosarcoma

Objective(s):To explore the value of circular RNA in the diagnosis and prognosis of osteosarcoma by Meta analysis.Circular RNA hsa＿circ＿0032462(circ SIPA1L1)was selected to explore the effect of hsa＿circ＿0032462 on the proliferation,migration and apoptosis of osteosarcoma,and to further explore the related mechanism of hsa＿circ＿0032462 regulation of osteosarcoma,so as to provide new research ideas for the diagnosis and treatment of osteosarcoma.Methods:1.Pub Med,Webof Science and Embase databases were used to collect studies on the relationship between circular RNA and the diagnosis and prognosis of osteosarcoma.The database was built until September 9,2022.Newcastle-Ottawa Scale was used to evaluate the quality of the literature.Diagnostic efficacy,overall survival and other related data were extracted and the bias risk was evaluated.Stata14.0 was used for Meta analysis.2.The specific circular RNAhsa＿circ＿0032462 was screened from GEO database.Then,the expression of hsa＿circ＿0032462 in 10 osteosarcoma and 10 osteochondroma patients and osteosarcoma cell lines was detected by q RT-PCR,and the localization of hsa＿circ＿0032462 in osteosarcoma cells was confirmed by FISH experiment.The cell models of hsa＿circ＿0032462 knock-down and overexpression were constructed by lentiviral vector.The effects of hsa＿circ＿0032462 on the proliferation,migration and apoptosis of osteosarcoma cells were detected by Transwell test,cell scratch test,CCK8 test,clone formation test and flow cytometry apoptosis test.Bioinformatics was used to predict the binding sites between hsa＿circ＿0032462 and mi R-411-5p and RAB9 A.The binding of hsa＿circ＿0032462and mi R-411-5p to RAB9 A was verified by RNA Pull-down experiment,double luciferase reporter gene test and q RT-PCR.The effects of spongified mi R-411-5p on the proliferation,migration and apoptosis of osteosarcoma cells were verified by Transwell test,wound healing test,CCK8 test,clone formation test and flow cytometry apoptosis test.The role of hsa＿circ＿0032462 in the growth of osteosarcoma was further proved by subcutaneous tumor formation in nude mice.Results:Meta analysis finally included 23 articles,all of which were published in 2018 Mel 2022,including 16 articles on prognosis,10 articles on diagnosis,and 3 articles on both diagnosis and prognosis.According to the NOS scale,23 articles were evaluated,the total NOS score was 4-6,the average was 5.35,and the median was 5.5,suggesting that the quality of the Meta analysis was higher.Prognostic value evaluation through the overall survival rate Meta analysis results showed that there was significant heterogeneity among the studies(I~2: 67.6% quotient 0.0001),using random effect model analysis,the results showed that circ RNA was significantly correlated with overallsurvival(HR=2.53,95%CI:1.99-3.22).The publication bias of the literature included in the diagnostic Meta analysis was asymmetrically tested by Deek’s funnel chart.The results showed that the trend of concentration was obvious and there was no publication bias(Prun0.98).The sensitivity,specificity,positive likelihood ratio,negative likelihood ratio and diagnostic odds ratio were 0.74(95%CI:0.63-0.83),0.82(95% 95%CI:8.39-0.87),4.21(95%CI:3.33-5.33),0.31(95%CI:0.21-0.45)and 13.51(95%CI:8.39-21.37)respectively.SROC showed that AUC was 0.86(95%CI:0.82-0.88).QRT-PCR assay showed that the expression of hsa＿circ＿0032462 was significantly increased in osteosarcoma tissues and cells,and FISH assay suggested that it was mainly located in the cytoplasm.Transwell test,cell scratch test,CCK8 test,clone formation test and flow cytometry apoptosis test proved that silent hsa＿circ＿0032462 could inhibit the proliferation and migration of osteosarcoma cells and promote their apoptosis.RNAPull-down assay,double luciferase reporter gene assay and flow cytometry apoptosis assay proved that hsa＿circ＿0032462 could control the proliferation,migration and apoptosis of osteosarcoma cells through sponge adsorption of mi R-411-5p.Transwell test,wound healing test,CCK8 test,clone formation test and flow cytometry apoptosis test proved that spongified mi R-411-5p could weaken the effect of hsa＿circ＿0032462 on the proliferation,migration and apoptosis of osteosarcoma cells.QRT-PCR assay and double luciferase reporter gene assay confirmed the binding of mi R-411-5p and RAB9 A.Western blotting and q RT-PCR experiments confirmed that hsa＿circ＿0032462 can control the proliferation,migration and apoptosis of OS through mi R-411-5p/RAB9 A axis.The subcutaneous tumorigenesis experiment of nude mice further proved the role of hsa＿circ＿0032462/mi R-411-5p/RAB9 A axis in the growth of osteosarcoma.Conclusion(s):1.Meta analysis showed that circular RNA was related to the prognosis of OS and could be used as a biomarker for predicting the prognosis of OS.2.Meta analysis suggest that circular RNA is related to the diagnosis of OS and can be used as a biomarker for auxiliary diagnosis of OS.3.hsa＿circ＿0032462 is up-regulated in OS tissues and cells,and is mainly located in the cytoplasm.4.Silencing hsa＿circ＿0032462 can inhibit the proliferation and migration of OS cells and promote the apoptosis of OS cells.5.hsa＿circ＿0032462 can control the proliferation,migration and apoptosis of OS cells by sponge adsorption of mi R-411-5p.6.Spongified mi R-411-5p could attenuate the effects of silence hsa＿circ＿0032462on the proliferation,migration and apoptosis of OS cells.7.hsa＿circ＿0032462 can control the proliferation,migration and apoptosis of OS through mi R-411-5p/RAB9 A axis.