MicroRNA-195 plays a tumor-suppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion

Abstract

Accumulating evidence has implicated the deregluation of miRNAs in tumorigenesis. Previous studies have reported that microRNA-195 (miR-195) is markedly down-regulated in human glioblastoma cells, compared with normal brain tissue, but the biological role of miR-195 in glioblastoma development is currently unknown. In this study, we define a tumor-suppressor role for miR-195 in human glioblastoma cells. Over-expression of miR-195 in glioblastoma cell lines robustly arrested cell cycle progression and significantly repressed cellular invasion. We identified E2F3 and CCND3 as functional downstream targets of miR-195 in glioblastoma cells. Through knockdown studies, we demonstrated that E2F3 was the dominant effector of miR-195-mediated cell cycle arrest and that CCND3 was a key mediator of miR-195-induced inhibition of glioblastoma cell invasion. Furthermore, we showed that p27(Kip1) was an important regulator downstream of CCND3 and that the accumulation of p27(Kip1) in the cytoplasm might be responsible for the miR-195-mediated cell invasion inhibition in glioblastoma cells. This work provides evidence for the initial mechanism by which miR-195 negatively regulates both the proliferation and invasion of glioblastoma cells, suggesting that the down-regulation of miR-195 might contribute to the malignant transformation of glioblastoma cells and could be a molecular signature associated with glioblastoma progression.

Fig. 1.

Northern blot detected miR-195 expression and miR-195 triggered G1 arrest in glioblastoma cell lines. A, The difference between miR-195 and other miR-16 family members was shown, and the expression signature was detected at 55°C to demonstrate the specificity of the Northern blot. B, Relative expression of endogenous miR-195 in human brain tissues and several glioblastoma cells (U87MG, LN-308, LN-229, A172, T98G, U251, LN-428, U373, and U138), as shown by Northern blot. The miR-195 level was normalized by the intensity of U6 in each sample. C, miR-195 arrested U87MG cells and LN-308 cells at the G1/S transition. Cells were treated with nocodazole 24 h posttransfection, and cell cycle distributions were detected 20 h later.

Fig. 2.

CCND3 and E2F3 are direct primary functional targets of miR-195. CCND1, CCND3, and E2F3 possess binding sites for miR-195 in their 3′UTRs (A), and luciferase assays indicated that miR-195 directly down-regulated the expression of CCND1, CCND3, and E2F3 (B). Relative luciferase values were normalized to cotransfections with the pcDNA6.2-control and the psiCHECK-2-control. Data represent the mean ± standard deviation from 2 separate determinations performed in triplicate. *** P < .001, using a 2-tailed t test. C, Western blotting shows the endogenous expression levels of CCND1, CCND3, and E2F3 after transfection with miR-195 or their cognate siRNA in glioblastoma cells. β-actin served as an internal control.

Fig. 3.

E2F3 is involved in miR-195-mediated G1/S transition arrest. Cell cycle distributions are detected in U87MG (A), LN-308 (B), LN-229 (C), A172 (D), and U251 (E) glioblastoma cells. Over-expression of miR-195 and knockdown of CCND1, CCND3, and E2F3 indicated that knockdown of E2F3 promotes the accumulation of cells at the G0/G1 stage and arrests cells at the G1/S transition in a similar manner to miR-195; however, knockdown of CCND3 did not inhibit cell cycle progression, except for A172 and U251 cells.

Fig. 4.

miR-195 impairs cell invasion through extracellular matrix substrates and promotes the expression of p27^Kip1 in U87MG, LN-308, and U251 cells. A, Over-expression of miR-195 and knockdown of CCND3 inhibit cell invasion through ECM in U87MG cells and LN-308 cells, except for U251 cells. Forty-eighthours posttransduction, cells were collected and seeded in 8-μm pore size transwell chambers. After 20 h, the cells on the bottom side of each chamber were fixed and stained. Rescue of miR-195 expression and knockdown of CCND3 up-regulated both total expression of p27^Kip1 (B) and cytoplasmic expression of p27^Kip1 (with the COOH-terminal) in U87MG and LN-308 cells (C).

Fig. 5.

miR-195 regulates cell cycle progression and cell invasion in glioblastoma cells. In glioblastoma cells, miR-195 is down-regulated. E2F3 and CCND3 protein levels inversely correlate with miR-195 expression in glioblastoma cell lines. Moreover, E2F3 and CCND3 are direct targets of miR-195. The down-regulation of E2F3 can suppress the transcription of cell cycle-related genes and can cause accumulation of cells in G1 phase and inhibition of cell cycle progression. However, the miR-195–mediated CCND3 suppression can up-regulate the expression of cytoplasmic p27^Kip1 and affect the expression of other proteins controlling cell migration, resulting in repression of glioblastoma cell invasion.