| Glioma,as a very common malignant tumor in the human central nervous system,features rapid growth,strong invasion,high mortality and high disability rates,accounting for approximately 60%of adult primary brain tumors.Although great advances have been made in comprehensive therapies of glioma,including radiotherapy,chemotherapy and surgery,the clinical management of glioma still faces many challenges.Therefore,it is necessary to study glioma progression at the molecular level and to identify new therapeutic approaches.Currently,with the deepening understanding and application of molecular targeted therapy for glioma,the role of long noncoding RNA(lncRNA)in glioma development has attracted clinical attention.LncRNAs have been suggested to be substantially involved in the development and progression of glioma.For example,lncRNA TUG1 was identified as a tumor suppressor to be downregulated in glioma tissues,and its expression is related to the pathological grading and prognosis closely.Overexpression of TUG1 may promote the apoptosis of glioma cells in vitro,and it has the potential to be applied for the treatment of glioma.Another group reported that overexpressed lncRNA H19 in glioma tissues was negatively linked to the glioma prognosis and that silencing H19 inhibited the growth of glioma cells by modulating miR-675.LncRNA OIP5-AS1 is important for controlling neurogenesis during development,because it is highly expressed in the nervous system.According to the study by Kim et al,OIP5-AS1,a competing endogenous RNA(ceRNA)for HuR,suppressed the growth of the cervical cancer cell line HeLa.However,limited data are available concerning the possible association between OIP5-AS1 and glioma.Meanwhile,as illustrated by the study,miR-410 acts on its target gene to effectively regulate the proliferation and invasion of glioma cells.Actually,miR-410 has been proven to specifically inhibit Wnt-7b expression,thereby influencing the proliferation and invasion of oral squamous cell carcinoma cells.This study was undertaken to investigate the regulatory effects of lncRNA OIP5-AS1 and miR-410 on glioma.To address this problem,we measured the expression of OIP5-AS1 and miR-410 in glioma tissues by qRT-PCR.The results showed that OIP5-AS1 expression was higher and miR-410 was lower in glioma tissues.The expression levels of OIP5-AS1 negatively correlated to levels of miR-410.MTT,flow cytometry,Transwell assays,and wound-healing assays were used to measure the biological characteristics of U87 glioma cells.Downregulation of OIP5-AS1 induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells.Silencing OIP5-AS1 reduced cell proliferation,invasion and migration of glioma U87 cells and led to depressed expression levels of Wnt-7b,p-?-catenin,GSK-3?-pS9,c-Myc and cyclin D1.Luciferase reporter assays confirmed a targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.Taken together,this research suggested that silencing OIP5-AS1 may specifically block the Wnt-7b/?-catenin pathway via targeted upregulating miR-410,thereby inhibiting growth,invasion and migration while promoting apoptosis in glioma cells.Part one Expression of OIP5-AS1 and miR-410 in glioma tissuesObjective:In order to research the relationship between the expression levels of OIP5-AS1 and miR-410,we compared the expression of OIP5-AS1and miR-410 in glioma tissues and normal tissues.Methods:From October 2016 to October 2017,tissue samples were obtained from 65 glioma patients who underwent surgical resection at the Department of Neurosurgery in our hospital;diagnoses were confirmed by the pathological examination.Patients had not received any other therapies prior to surgery.Based on the pathological classification of tumors of the central nervous system published by WHO in 2007.Non-neoplastic brain tissue samples were obtained from 10 adult patients.The expression levels of OIP5-AS1 and miR-410 in glioma and normal tissues were measured by qRT-PCR.To understand the relationship between OIP5-AS1 and miR-410 by Starbase 2.0.All data were processed and analyzed by using the statistics software SPSS 20.0.Results:1.Clinical data of patients and sample providers.Tumor group:The patients had a mean age of 46.23±12.58 years,including 40 males and 25 females.There were 29 cases of low-grade glioma(grade I/II),including 12 cases of grade I and 17 cases of grade II,and 36 cases of high-grade glioma(grade III/IV),including 19 cases of grade III and 17 cases of grade IV.Control group:Non-neoplastic brain tissue samples were obtained from 10 adult patients.2.The expression levels of OIP5-AS1 were upregulated,but the expre-ssion levels of miR-410 were downregulated in glioma tissues compared with normal tissues.3.Expression levels of OIP5-AS1 and miR-410 were significantly associated with the pathological grading of glioma,because OIP5-AS1 was notably more highly expressed in high-grade glioma tissues than in low-grade tissues;however,miR-410 levels were lower in high-grade glioma than in low-grade tissues.According to the correlation analysis results,expression levels of OIP5-AS1 negatively correlated to levels of miR-410.OIP5-AS1 is a molecular sponge that regulates miR-410,as predicted by Starbase 2.0.Conclusion:1.The expression levels of OIP5-AS1 were upregulated,but the expre-ssion levels of miR-410 were downregulated in glioma tissues,they may affect the incidence of glioma.2.Expression levels of OIP5-AS1 and miR-410 were significantly asso-ciated with the pathological grading of glioma,and the expression levels of OIP5-AS1 negatively correlated to levels of miR-410.These results suggested that there maybe a targeting relationship between OIP5-AS1 and miR-410.Part two The effect of OIP5-AS1 and miR-410 on proliferation,invasion,migration and apoptosis of glioma cellsObjective:U87 cell lines that transfected with OIP5-AS1 siRNA and miR-410 inhibitors were established to investigate the effects of OIP5-AS1and miR-410 on the proliferation,invasion,migration and apoptosis of glioma cells.Methods:Cells were assigned into five groups:Blank group(cells without any transfection),NC group(cells transfected with NC sequence),OIP5-AS1siRNA group(cells transfected with OIP5-AS1 siRNA),miR-410 inhibitors group(cells transfected with miR-410 inhibitors),and OIP5-AS1siRNA+miR-410 inhibitors(siRNA+inhibitors)group(cells transfected with OIP5-AS1 siRNA and miR-410 inhibitors).Lipofectamine-2000(Invitrogen)was utilized for the transfection of U87 cells according to the manufacturer's protocol.Cell proliferation assessed by MTT assay,cell cycle and apoptosis evaluated by flow cytometry and Annexin V-FITC/PI,cell invasion detected by Transwell assay,cell migration measured by wound-healing assay.Results:1.Proliferation was apparently inhibited in the OIP5-AS1 siRNA group and was remarkably enhanced in the miR-410 inhibitors group compared with cells in the Blank group.In comparison with the OIP5-AS1 siRNA group,the siRNA+inhibitors group increased dramatically in terms of proliferation.2.OIP5-AS1 siRNA induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells,and the effect is reversed by miR-410 inhibitors.Compared with cells in the Blank group,those in the OIP5-AS1 siRNA group showed a significantly elevated proportion of G0/G1 phase,a reduced proportion of S phase,and apparently increased apoptosis rates.Cells in the miR-410 inhibitors group were significantly reduced in terms of proportion of G0/G1 phase cells and apoptosis rates,with elevated proportions of S phase cells.By comparison with cells in the OIP5-AS1 siRNA group,those in the siRNA+inhibitors group showed substantially fewer cells in the G0/G1 phase and lower apoptosis rates,with an increased proportion of S phase cells.No significant differences were found in terms of the proportion of G2 phase in all groups.This indicated OIP5-AS1 siRNA induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells.The effect is reversed in miR-410 inhibitors group.3.OIP5-AS1 siRNA reduced cell invasion and migration of glioma U87cells,while miR-410 inhibitors promote cell invasion and migration.The number of invasive cells and the wound-healing rate were substantially lower in the OIP5-AS1 siRNA group and significantly higher in the miR-410 inhibitors group.Furthermore,the siRNA+inhibitors group was statistically higher than the OIP5-AS1 siRNA group with respect to these two indexes.Conclusion:1.Silencing OIP5-AS1 reduced cell proliferation,invasion and migration of glioma U87 cells.2.Downregulation of OIP5-AS1 induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells.3.The miR-410 inhibitors reversed the inhibitory effect of OIP5-AS1siRNA on proliferation,invasion and migration.Part three The mechanism of OIP5-AS1 targets Wnt-7b/?-catenin via modulation of miR-410Objective:To investigate the mechanism of OIP5-AS1 targets Wnt-7b/?-catenin via modulation of miR-410,and to clarify the targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.Methods:The experimental groups as same as Part two.Expression of Wnt-7b/?-catenin signaling pathway-related proteins was measured by Western blotting analysis.The targeting relationship among miR-410,OIP5-AS1 and Wnt-7b was detected by Dual-luciferase reporter assay.Results:1.The protein expression levels in U87 glioma cells.The expression levels of total GSK-3?and total?-catenin were not significantly different in all groups.The protein expression levels of Wnt-7b,p-?-catenin,GSK-3?-pS9,c-Myc and cyclin D1 were lower in the OIP5-AS1siRNA group,but were remarkably higher in the miR-410 inhibitors group compared with the Blank group.Moreover,cells in the siRNA+inhibitors group were dramatically lower in terms expression of miR-410 and increased in terms of expression of Wnt-7b/?-catenin pathway-related proteins when compared to cells in the OIP5-AS1 siRNA group.2.There was a targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.With the use of online prediction software Target Scan,we identified the binding site between Wnt-7b and miR-410.According to the dual-luciferase activity assay,miR-410 mimics did not significantly differ from the NC group with respect to the effect on the luciferase activity of OIP5-AS1-mt and Wnt-7b-mt;however,miR-410 mimics significantly downregulated the luciferase activity of wild-type OIP5-AS1–wt and Wnt-7b-wt.These results suggested that there was a targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.Conclusion:1.Silencing OIP5-AS1 reduced cell proliferation,invasion and migration of glioma U87 cells and led to depressed expression levels of Wnt-7b,p-?-catenin,GSK-3?-pS9,c-Myc and cyclin D1.2.The miR-410 inhibitors up-regulated the expression of Wnt-7b and activated the Wnt signaling pathway and the expression of related protein.3.There was a targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.Part four Effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivoObjective:To observe the effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivo.Methods:Thirty-two male BALB/c nude mice were assigned into four groups with 8 per group.Mice were injected subcutaneously with cell suspensions(approximately 5×10~7cells/ml)in the Blank,NC,OIP5-AS1siRNA and miR-410 inhibitors groups.After formation of tumors,both length(a)and width(b)of the tumor bodies were measured with Vernier calipers,and tumor volume was calculated based on the formula:V=(ab~2)/2.The growth curves of tumors were drawn accordingly.Three weeks after tumor inoculation,the mice were sacrificed by cervical dislocation.Tumor tissues were obtained,and tumor weights were measured.The tumor tissues obtained from mice were then stained with hematoxylin and eosin(HE).Results:The nude mice in the NC group were not significantly different in terms of tumor volume or weight at various time points compared with those of the Blank group.For the nude mice in the OIP5-AS1 siRNA group,their tumor growth was inhibited with significantly reduced tumor volume and weight when compared with mice in the Blank and NC groups;the miR-410 inhibitors group was completely opposite to the OIP5-AS1 siRNA group.HE staining of tumors in the Blank and NC groups showed typical histological features of glioma,including high cellular density and an infiltrative growth pattern;the changes were substantial in miR-410 inhibitors group.In the OIP5-AS1 siRNA group,necrotic foci were observed,tissue alignment became irregular,and glioma cellular density decreased significantly compared with the other three groupsConclusion:Silencing OIP5-AS1 will inhibited the growth of glioma in nude mice,while inhibiting the expression of miR-410 promoted the growth of glioma in nude mice.Summary:1.The expression levels of OIP5-AS1 were upregulated,but the expre-ssion levels of miR-410 were downregulated in glioma tissues,2.Silencing OIP5-AS1 reduced cell proliferation,invasion and migration,while promoted the cellular apoptosis of glioma cells.Inhibition of miR-410reversed the effect of OIP5-AS1 siRNA.3.There was a targeting relationship between OIP5-AS1 and miR-410,as well as between miR-410 and Wnt-7b.Silencing OIP5-AS1 specifically block the Wnt-7b/?-catenin pathway via targeted upregulating miR-410.The miR-410 inhibitors up-regulated the expression of Wnt-7b and activated the Wnt signaling pathway and the expression of related protein.4.In nude mice,OIP5-AS1 knockdown inhibited tumor growth,while inhibiting the expression of miR-410 promoted the growth of glioma. |
PDF Full Text Request