LCN2-derived Circular RNA,hsa_circ₀088732,inhibits Cell Apoptosis And Promotes EMT Process Through MiR-661/RAB3D Axis In Glioma

Gliomas are the most common malignant tumors of the central nervous system.Low-grade gliomas have a good prognosis.High-grade gliomas are called glioblastomas（GBM）and are the most aggressive malignant primary brain tumor.For GBM patients,the main treatment is surgical resection,supplemented by radiation therapy and chemotherapy.Despite this,the median survival is only 12-15 months.The expression of LCN2 was up-regulated in glioma.CircRNA has been a hot topic in tumor research in recent years.A large number of studies have shown that circRNA plays an important role in the occurrence and development of many tumors.At present,the pathogenesis of gliomas has not been fully elucidated,and further exploration is urgently needed to find more therapeutic targets and targeted drug development.Methods:1.CircRNAs related to LCN2 were retrieved through the database.FISH experiments were used to screen the highest abundance of circRNAs in the collected glioma cancer tissues for subsequent experiments.Candidate circRNAs were identified by PCR amplification,RNase R resistance,and next-generation sequencing methods.The circRNA-targeting siRNA was designed and synthesized to transfect cell lines LN229 and U87-MG,and the relevant indicators of apoptosis and EMT process were detected.2.Through methods such as database,qPCR,and double luciferase experiments,miRNAs interacted directly with circRNA were screened,and miRNA expression was further intervened in cell lines to detect indicators of apoptosis and EMT.The target of miRNA was verified by online database,Western blot and double luciferase experiments,and the target of miRNA was interfered in tumor cells to detect apoptosis and related indexes of EMT process.Finally,a rescue experiment was performed to detect the apoptosis-related and EMT-related indicators by interfering with circRNA,miRNA,and miRNA targets in cells.3.The circRNA low-expressing and over-expressing LN229 cell lines were constructed and injected into nude mice to form subcutaneous tumors,and tumor growth was continuously monitored.The expression levels of circRNA,miRNA and downstream targets of miRNA were detected by qPCR and/or western blot.Immunohistochemistry,Tunel and HE staining were used to assess tumor cell proliferation,apoptosis and pathology.Results:1.Hsa_circ₀088732 has the highest abundance in glioma tissues and is located in the cytoplasm.The expression level of glioma tissues was significantly up-regulated compared with adjacent tissues.Interfering with hsa_circ₀088732 in vitro can up-regulate tumor cell apoptosis and inhibit EMT characteristics.2.a.qPCR results showed that the expression level of miR-661 in glioma tissues was significantly lower than adjacent tissues,while miR-7 had no expression difference.RAB3D is a downstream target of miR-661,and its expression is related to glioma development.b.The double luciferase experiment results showed that hsa_circ₀088732 can directly interact with miR-661,and RAB3D can be used as a direct target of miR-661.In vitro experiments,interfering with miR-661 expression could promote tumor cell survival and EMT characteristics,while interfering with RAB3D expression had opposite effects on tumor cells.c.Rescue experiments were performed in glioma cell lines,and the results showed that hsa_circ₀088732/miR-661/RAB3D signal axis can critically regulate glioma cell apoptosis and EMT process.3.The over expression of hsa_circ₀088732 could promote the tumorigenesis of malignant cells,accompanied by the weakened apoptosis signal and increased proliferation.The low expression of hsa_circ₀088732 could inhibit tumorigenesis,causing increased apoptosis and weakened proliferation of glioma cells.In glioma tissue,hsa_circ₀088732 overexpression was accompanied by a decrease in miR-661 expression,while RAB3D expression was up-regulated.Conclusion:1.The expression of hsa_circ₀088732 has a positive correlation with the occurrence of glioma,and can promote the occurrence and development of glioma.Intervention of hsa_circ₀088732 expression can inhibit glioma growth and promote cell apoptosis.2.By adsorbing miR-661,hsa_circ₀088732 can promotes the occurrence and development of glioma,and indirectly promotes the expression of RAB3D,and then affects tumor cell apoptosis and EMT process.3.The miR-661 has the function of inhibiting the development of glioma.It is mainly through regulating its downstream target RAB3D,inhibiting its expression,and regulating tumor cell apoptosis and EMT process.