MicroRNA-370 suppresses the progression and proliferation of human astrocytoma and glioblastoma by negatively regulating β-catenin and causing activation of FOXO3a

Abstract

Certain microRNAs (miRs) regulate the progression and metastasis of various cancer types. In the present study, the role of miR-370 in the progression and proliferation of human astrocytoma and glioblastoma cells was assessed and the underlying molecular mechanism was investigated. miR-370 levels in clinical specimens of human glioma and peritumoral tissues were determined by reverse-transcription quantitative PCR. Oligonucleotide mimics and inhibitors were transfected into the U-251MG human astrocytoma cell line and the and U-87MG glioblastoma cell line and the cell viability of was determined by an MTT assay. The expression of β-catenin and forkhead box protein (FOX)O3a was determined by western blot analysis. The results revealed that the expression of miR-370 in human glioma tissues was significantly decreased compared with that in peritumoral tissues. The miR-370 levels in patients with grade III/IV gliomas were significantly decreased compared with those in grade I/II. Transfection with miR-370 mimics inhibited the proliferation of U-251MG and U-87MG cells. Furthermore, the miR-370 levels were negatively correlated with β-catenin and positively correlated with nuclear FOXO3a. In conclusion, miR-370 inhibited the proliferation of human glioma cells by regulating the levels of β-catenin and the activation of FOXO3a, suggesting that miR-370 was a tumor suppressor in the progression of human astrocytoma and glioblastoma cells.

Keywords: forkhead box protein O3a; gliomas; microRNA-370; β-catenin.

Figure 1.

(A) miR-370 levels in 8 clinical specimens of glioma and paired peritumoral tissues as determined by reverse-transcription quantitative polymerase chain reaction. miR-370 levels in glioma tissues were downregulated compared with those in peritumoral tissues. **P<0.01. (B) miR-370 expression in grade I/II and grade III/IV gliomas as well as paired peritumoral tissue was negatively associated with the glioma grade. Values are expressed as the mean ± standard deviation (n=8 per group). **P<0.01 and ***P<0.001, compared with peritumoral tissues.^#P<0.05, compared with grade I/II. miR, microRNA; Ctrl., control (peritumoral tissue).

Figure 2.

miR-370 expression is decreased in U-87MG human glioblastoma and U-251MG astrocytoma cell lines as well as in astrocytes (normal controls). The levels of miR-370 were detected by reverse-transcription quantitative polymerase chain reaction. **P<0.01, compared with astrocytes. miR, microRNA.

Figure 3.

Aberrant expression of β-catenin and FOXO3a in human glioma and peritumoral tissues as determined by western blot analysis. (A) Western blot images of three pairs of glioma (grade I/II) and peritumoral tissues are presented. (B) Quantified expression levels of β-catenin and FOXO3a. **P<0.01 compared with paired peritumoral tissues. Ctrl., control; FOX, forkhead box protein.

Figure 4.

Transfection with miR-370 mimics suppresses the proliferation of (A) the U-251MG astrocytoma cell line and (B) the U-87MG human glioblastoma cell line. The viability of negative control or miR-370 mimics-transfected cells was determined by an MTT assay at 1, 2, 3, 4 and 5 days. *P<0.05, **P<0.01, compared with negative control group. miR, microRNA; Ctrl., control; d, days.

Figure 5.

Transfection of miR-370 mimics inhibits β-catenin and c-myc expression in U-251MG cells. (A) Cells were transfected with miR-370 mimics or negative controls for 48 h. The levels of β-catenin and c-myc were determined by western blot analysis. (B) The ratios of β-catenin and c-myc were presented in a bar graph. **P<0.01, compared with the negative control group. miR, microRNA; Ctrl., control.

Figure 6.

Transfection of miR-370 mimics leads to a decrease of phosphorylated FOXO3a and accumulation of FOXO3a in the nuclei of U-251MG cells. Cells were transfected with miR-370 mimics or negative controls for 48 h. (A) The levels of phosphorylated FOXO3a and total FOXO3a were detected by western blot analysis. β-actin was used as internal reference. (B) Nuclear FOXO3a was determined by western blot analysis in miR-370 mimics- and negative control-transfected cells. Lamin B1 was used as internal reference gene in the experiment. Ctrl., control; FOX, forkhead box protein; miR, microRNA.