A study of regulating mechanisms of miRNA-451 in the epithelial-mesenchymal transition of human glioma cells

Abstract

Objective To investigate the mechanism of miRNA-451 overexpression in the regulation of epithelial-mesenchymal transition(EMT) in human glioma cells through target calcium binding protein 39 (CAB39) and to explore the effects of miRNA-451 overexpression on invasion and migration of glioma cells. Methods The U251 glioma cells were cultured and divided into 3 groups including control group, nonsense sequence group and miRNA-451 mimics treatment group (briefly miRNA-451 group). The expression of miRNA-451 was detected by RT-PCR in glioma cells after transfection. The expression proteins of CAB39, E-cadherin, N-cadherin, Vimentin, TWIST, MMP-2, MMP-9, Snail, AKT, P-AKT were detected by Western blot. Cell immunofluorescence assay was used to further validate the expression levels of N-cadherin and Vimentin. The wound healing and transwell experiments were performed to detect changes in cell invasion and migration abilities. Results The miRNA-451 expression level in the miRNA-451 group was significantly higher compared with those in the control and nonsense sequence groups (both P<0.05). The results of Western blot revealed that the expression levels of CAB39, N-cadherin, Vimentin, TWIST, MMP-2, MMP-9, Snail, P-AKT in miRNA-451 group were decreased (all P<0.05) and the expression level of E-cadherin was increased as compared with those in the control and nonsense sequence groups (all P<0.05). Cell immunofluorescence assay showed that the relative expressions of N-cadherin and Vimentin in miRNA-451 group were significantly lower than those in control group (both P<0.05). The results of the wound healing showed that the scar repair rate of miRNA-451 group was significantly lower than those in the control and nonsense groups at 24 hrs or 48 hrs post would healing(all P<0.05). The number of cells in the miRNA-451 group was significantly decreased as compared with the other two groups at 48 hrs post transfection (both P<0.05). Conclusion The regulation of PI3K/AKT pathway by miRNA-451 might be through the decrease of CAB39 expression, which could cause the inhibition of EMT in human glioma cells and their invasion and migration. Key words: MicroRNAs; Glioma; Epithelial-mesenchymal transition; Neoplasm invasiveness; miRNA-451