Investigation Of The Role Of Circular Non-coding RNA Hsa＿circ＿0059511 In Promoting Glioma Progression And The Potential Molecular Mechanism Related To Its Function

background: Glioma is a malignant tumor originating from glial cells,which is the most common primary intracranial tumor in neurosurgery.Patients with low-grade gliomas usually can have a good prognosis,while most high-grade gliomas patients usually have a poor prognosis.The five-year survival of the patient with high-grade glioma still remain low up to now.Studies in recent years have shown that circular RNA(circ RNA)plays an important role in the development of various tumors.It hsa been found that hsa＿circ＿0059511 expressed in the glioblastoma tissue of secondary temozolomideresistance,suggesting that it may have the effect of an onogene.However,the role of hsa＿circ＿0059511 in gliomas remains unknown;As we know,the research that the association between hsa＿circ＿0059511 and glioma hsa not been studied hsa farTherefore,the purpose of this study is to explore the influence of hsa＿circ＿0059511 on malignant biological behavior of glioma and to explore the potential molecular mechanism of its function,hsa as to lay a new target for the precise treatment of glioma.Method:1.Real-time fluorescence quantitative PCR(q RT-PCR)was used to detect the expression of hsa＿circ＿0059511 in glioma tissues and glioma cell lines.Fluorescence in situ hybridization(FISH)was used to verify the expression and localization of hsa＿circ＿0059511in cells.Lentiviral sh RNA vector against hsa＿circ＿0059511 and pc DNA3.1-hsa＿circ＿0059511 overexpression vector were constructed and transfected into glioma U251 cells and T98 G cell lines.U251 cells and T98 G cells with stable low expression and overexpression of hsa＿circ＿0059511 were screened by puromycin and G418 respectively.Cell proliferation was determined by CCK-8 and EDU assay,cell growth were determined by plate cloning test,cell migration and invasion were determined by Transwell assay to explore the effects of down-regulation and up-regulation of hsa＿circ＿0059511 expression on the malignant physiology of glioma cells.2.Through the RDB data library https://circinteractome.nia.nih.gov of online circ RNA and micro RNA and http://www.mirdb.org/ microrna predicting the downstream target micro RNA of hsa＿circ＿0059511.It is shown that hsa-mi R-194-5 may be the downstream target micro RNA of hsa＿circ＿0059511.The targeted binding and binding sites were verified by double luciferase reporter gene assay,and the expression and function of hsa-mi R-194-5p in U251 and T98 G cell lines after silencing or overexpression of hsa＿circ＿0059511 were detected by QRT-PCR.Cell transfection assay was used to detect the changes of cell growth after overexpression and inhibition of hsa-mi R-194-5 expression in wild type cells,sh RNA negative control cells and cells with low expression of hsa＿circ＿0059511,respectively.3.The lentivirus sh RNA-0059511 was stably transfected into the 251 cells and were then injected into the nude mice subcutaneously to form the subcutaneously transplanted tumors by the way of the injection of the cells.Followed,tumor growths were monitored.The expression level of circ RNA in the tumor was detected by q RT-PCR assay,and the tumor tissue was confirmed by HE staining after the mice had been injected the cells for about 45 days,and at that time,the mice had been euthanasia.Results:1.Compared with paired peri-cancerous tissues,hsa＿circ＿0059511 was overexpressed in 34 of 43 gliomas(p < 0.01).Compared with the expression of SVGP12 in noncancerous astrocytes,hsa＿circ＿0059511 was overexpressed in 5 glioma cell lines: U87 MG,T98G,U251,A172 and LN229(p <0.05).Fish fluorescence in situ hybridization analysis showed that the circ RNA was mainly located in the cytoplasm of the glioma cells.The decrease of hsa＿circ＿0059511 decreased whereas overexpression of hsa＿circ＿0059511 increased the proliferation,migration and invasion of U251 and T98 G cells.2.Through the double luciferase reporter gene experiment,we found that the wild type hsa＿circ＿0059511 could bind with hsa-mi R-194-5p,resulting in the decrease of luciferase activity,while the hsa＿circ＿0059511 mutant with deletion of hsa-mi R-194-5p binding site could not bind to the micro RNA.Functional studies showed that silencing hsa＿circ＿0059511 could increase the expression of hsa-mi R-194-5p,while overexpressing hsa＿circ＿0059511 could decrease the expression of hsa-mi R-194-5p.More importantly,the transfection of hsa-mi R-194-5p inhibitor can reverse the decline of the fine cell growth induced by silent hsa＿circ＿0059511.The results show that there is a possibility of interaction between hsa＿circ＿0059511 and hsa-mi R-194-5p,and the micro RNA hsa-mi R-194-5p may be the downstream target micro RNA of hsa＿circ＿0059511.3.Injection of U251 cells can induce the growth of transplanted tumor in nude mice.The low expression of hsa＿circ＿0059511 can inhibit the growth of tumor.The results of q RT-PCR showed that injection of lentivirus sh RNA-0059511 could inhibit not only the expression of hsa＿circ＿0059511,but also the growth of transplanted tumor in nude mice.Compared with U251 cells injected with sh RNA negative control,the difference was statistically significant(p < 0.05).Conclusion:1.There is a positive correlation between hsa＿circ＿0059511 and the development of glioma,which can promote the development of glioma.Pre-treatment of hsa＿circ＿0059511expression can inhibit tumor growth and progression.2.hsa＿circ＿0059511 targeted binding of hsa-mi R-194-5p;hsa-mi R-194-5p may be the downstream target micro RNA of hsa＿circ＿0059511..3.Silencing hsa＿circ＿0059511 significantly inhibited the growth of nude mice transplanted tumor.