MiR-15b targets cyclin D1 to regulate proliferation and apoptosis in glioma cells

Abstract

Aim: To investigate the role and mechanism of miR-15b in the proliferation and apoptosis of glioma.

Methods: The miR-15b mimics were transfected into human glioma cells to upregulate the miR-15b expression. Cyclin D1 was determined by both western blotting analysis and luciferase reporter assay. Methylthiazol tetrazolium (MTT) and flow cytometry were employed to detect the cell proliferation, cell cycle, and apoptosis.

Results: Overexpression of miR-15b inhibits proliferation by arrested cell cycle progression and induces apoptosis, possibly by directly targeting Cyclin D1. Both luciferase assay and bioinformatics search revealed a putative target site of miR-15b binding to the 3'-UTR of Cyclin D1. Moreover, expression of miR-15b in glioma tissues was found to be inversely correlated with Cyclin D1 expression. Enforced Cyclin D1 could abrogate the miR-15b-mediated cell cycle arrest and apoptosis.

Conclusions: Our findings identified that miR-15b may function as a glioma suppressor by targeting the Cyclin D1, which may provide a novel therapeutic strategy for treatment of glioma.

Figure 1

(a) MiR-15b expression in glioma tissues and normal brain specimens. The relative levels of miR-15b were measured by real-time PCR assay. (b, c) MiR-15b expression increased about 5.02-fold and 3.98-fold, at 48 h after transfection of miR-15b mimics in U87 and LN229 cells, respectively. Total RNA was extracted using Trizol reagent. The relative expression of miR-15b was calculated by using a 2⁻ΔΔCt method. The data are presented as the mean ± SD. **P < 0.05 compared to the control.

Figure 2

Cyclin D1 is a direct target of miR-15b in glioma cells. (a) Bioinformatics analysis of the predicted interactions of miR-15b with the binding sequence at the 3′UTR of Cyclin D1 mRNA. (b) Overexpression of miR15b downregulates endogenous Cyclin D1 expression level in U87 and LN229 glioma cells. (c, d) Luciferase assay showed a reduction of luciferase expression following the cotransfection of pGL3-Cyclin D1 vector together with miR-15b. The data were normalized by the ratio of firefly and Renilla luciferase activities. The data are presented as the mean ± SD. **P < 0.05 compared to the control.

Figure 3

Negative link between miR-15b and Cyclin D1 expression in glioma tissues. (a) The relative expression of Cyclin D1 was measured by real-time PCR assay. (b) Inverse correlation of miR-15b expression with Cyclin D1 mRNA expression using Pearson's correlation analysis.

Figure 4

MiR-15b suppresses the growth of glioma cells. (a) MTT assay reveals a significantly inhibitory effect of miR-15b mimics treated cell (Student's t-test). (b) Overexpression of miR-15b results in the cell cycle arrest at G0/G1 phases in glioma cells. (c) MiR-15b induced apoptosis in both U87 and L229 glioma cells (Student's t-test). The data are presented as the mean ± SD. **P < 0.05 compared to the control.

Figure 5

Cyclin D1 plays a crucial role in the suppressive proliferative process of miR-15b in glioma cells. (a) Western bolt analysis displays that ectopic of Cyclin D1 abrogates the miR-15b-mediated Cyclin D1 expression partly. (b) Ectopic expression of Cyclin D1 counteracts the G1 arrest induced by miR-15b in glioma cells. The representative results out of three times of experiments were shown here.