Objective:To discuss the effects of microRNA(miR)-199a-5p overexpression on cell migration and apoptosis in the glioblastoma U251 cells,and to clarify the targeting regulatory relationship between miR-199a-5p and caveolin-1(CAV-1).Methods:The glioblastoma U251 cells and oligodendroglioma Hs683 cells were cultured in vitro.Western blotting method was used to detect the expression levels of CAV-1 protein in 2 kinds of cells;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-199a-5p in 2 kinds of cells.The U251 cells were divided into blank group(non-transfection),mimics NC group(transfected with empty vector),and miR-199a-5p mimics group(transfected with miR-199a-5p mimics).The Hs683 cells were divided into blank group(no transfection),inhibitor NC group(transfected with empty vector),and miR-199a-5p inhibitor group(transfected with miR-199a-5p inhibitor).RT-qPCR method was used to detect the transfection efficiency of the cells in various groups;Western blotting method was used to detect the expression levels of CAV-1 protein in the cells in various groups.TargetScan database was used to predict the binding sites between miR-199a-5p and CAV-1 in the 3'untrans lated region(3'UTR);psiCHECKTM-2-CAV-1-WT and psiCHECKTM-2-CAV-1-Mut were co-transfected with miR-199a-5p mimics and mimics NC into the U251 cells,respectively,forming psiCHECKTM-2-CAV-1-WT+mimics NC group,psiCHECKTM-2-CAV-1-WT+miR-199a-5p mimics group,psiCHECKTM-2-CAV-1-Mut+mimics NC group,and psiCHECKTM-2-CAV1-Mut+miR-199a-5p mimics group;dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-199a-5p and CAV-1;cell scratch assay was used to detect the scratch healing rates of the U251 cells in various groups;flow cytometry was used to detect the apoptotic rates of the U251 cells in various groups.Results:The Western blotting and RT-qPCR results showed that compared with Hs683 cells,the expression level of CAV-1 protein in the U251 cells was significantly decreased(P<0.05);compared with U251 cells,the expression level of miR-199a-5p in the Hs683 cells was significantly increased(P<0.01).Compared with blank group and mimics NC group,the expression level of miR-199a-5p in the U251 cells in miR-199a-5p mimics group was significantly increased(P<0.01),the expression level of CAV-1 protein in the U251 cells in miR-199a-5p mimics group was significantly decreased(P<0.05).Compared with blank group,the expression levels of miR-199a-5p in the Hs683 cells in inhibitor NC group and miR-199a-5p inhibitor group were significantly decreased(P<0.01).No significant differences were observed in the expression levels of CAV-1 protein in the Hs683 cells among various groups(P>0.05).The dual-luciferase reporter gene assay results showed that psiCHECKTM-2-CAV-1-wild type(WT)and psiCHECKTM-2-CAV-1-mutant(Mut)expression vectors were successfully constructed;compared with psiCHECKTM-2-CAV-1-WT-mimics NC group,the relative luciferase activity of WT CAV-1 in the U251 cells in psiCHECKTM-2-CAV-1-WT-miR-199a-5p mimics group was significantly decreased(P<0.01).The cell scratch assay results showed that at 12,24,and 48 h after transfection,compared with blank group,the scratch healing rate of the U251 cells in miR-199a-5p mimics group was significantly decreased(P<0.05 or P<0.01).The flow cytometry results showed that compared with blank group and mimics NC group,the apoptotic rate of the U251 cells in miR-199a-5p mimics group was significantly increased(P<0.01).Conclusion:Transfection of mature miR-199a-5p mimics into the glioblastoma U251 cells can reduce the expression of CAV-1 protein,inhibit glioma cell migration,promote apoptosis,and suppress tumorigenesis and development.The targeting relationship between miR-199a-5p and CAV-1 may represent a potential mechanism for glioma development and could serve as a potential diagnostic and therapeutic target for glioma.

Objective To investigate the ameliorative effect of deferoxamine(DFO)on cognitive impairment in a rat model of type 1 diabetes mellitus(T1DM)and to elucidate the molecular mechanisms underlying cerebral iron overload in T1DM rats.Methods Thirty-six healthy male SD rats(weighing 180～250 g)were randomly assigned into a blank control group(Ctrl),a T1DM model group and a DFO group,with 12 rats in each group.A single dose of 65 mg/kg streptozotocin(STZ)was intraperitoneally injected to the rats to establish a T1DM model,and those with fasting blood glucose≥16.7 mmol/L at 3 d later were designated as the T1DM group.Intracerebroventricular administration of DFO(5 μg/kg·d)was given to the DFO group for 28 consecutive days since 21 d after STZ injection.Morris water maze test was carried out to assess the spatial learning and memory abilities.Nissl staining and immunofluorescence assay were applied to observe neuronal morphology and number in the hippocampus and cortex.The iron content in the hippocampus was measured with inductively coupled plasma mass spectrometry(ICP-MS).The expression levels of iron metabolism related proteins were detected with Western blotting.Results In the T1DM group,significant declines in learning and memory abilities(P<0.01)and impaired neuronal morphology and reduced neuronal counts in the hippocampal CA1 region(P<0.01),CA3 region(P<0.01),and cortex(P<0.05)were observed when compared with those in the Ctrl group.ICP-MS analysis showed a marked increase in the hippocampal iron content in the T1DM group(P<0.01).Western blot results demonstrated that T1DM rats exhibited obviously up-regulated expression of iron storage proteins FTH and FTL in both the hippocampus(P<0.01,P<0.05)and the cortex(P<0.05,P<0.01),enhanced expression of iron import protein DMT1 in both the hippocampus and the cortex(P<0.05),while decreased expression of iron export protein FPN1 in the hippocampus(P<0.01)and the cortex(P<0.05).DFO treatment significantly ameliorated all above abnormalities.Conclusion The declines in learning and memory in T1DM rats are closely associated with neuronal damage induced by cerebral iron overload.Iron import protein DMT1 and export protein FPN1 jointly regulate cerebral iron content in T1DM rats.DFO reduces brain iron levels and mitigates iron overload-mediated neuronal injury by modulating the expression of DMT1 and FPN1.