Abstract:

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.

Key words: Glioma cells, MicroRNA-199a-5p, Caveolin-1, Cell migration, Apoptosis