MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes

Abstract

MicroRNA-34a (miR-34a) is a transcriptional target of p53 that is down-regulated in some cancer cell lines. We studied the expression, targets, and functional effects of miR-34a in brain tumor cells and human gliomas. Transfection of miR-34a down-regulated c-Met in human glioma and medulloblastoma cells and Notch-1, Notch-2, and CDK6 protein expressions in glioma cells. miR-34a expression inhibited c-Met reporter activities in glioma and medulloblastoma cells and Notch-1 and Notch-2 3'-untranslated region reporter activities in glioma cells and stem cells. Analysis of human specimens showed that miR-34a expression is down-regulated in glioblastoma tissues as compared with normal brain and in mutant p53 gliomas as compared with wild-type p53 gliomas. miR-34a levels in human gliomas inversely correlated to c-Met levels measured in the same tumors. Transient transfection of miR-34a into glioma and medulloblastoma cell lines strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion, but transfection of miR-34a into human astrocytes did not affect cell survival and cell cycle status. Forced expression of c-Met or Notch-1/Notch-2 transcripts lacking the 3'-untranslated region sequences partially reversed the effects of miR-34a on cell cycle arrest and cell death in glioma cells and stem cells, respectively. Also, transient expression of miR-34a in glioblastoma cells strongly inhibited in vivo glioma xenograft growth. Together, these findings represent the first comprehensive analysis of the role of miR-34a in gliomas. They show that miR-34a suppresses brain tumor growth by targeting c-Met and Notch. The results also suggest that miR-34a could serve as a potential therapeutic agent for brain tumors.

Figure 1. miR-34a inhibits the expression of multiple oncogenes and binds to their 3′UTR in brain tumor cells

A) Glioma cells and stem cells, medulloblastoma cells or astrocytes were transfected with either pre-miR-34a or pre-miR-con for 72 hrs. c-Met, Notch-1, Notch-2 and CDK6 protein levels were measured by immunoblotting. The results show that miR-34a expression downregulates c-Met, Notch-1, Notch-2 and CDK6 protein levels (right panels). The predicted seed matching between miR-34a and the oncogenes' 3′UTR sequences is shown in the left panels. B) Glioma cells were transfected with pre-miR-34a or pre-miR-con for 24 hrs prior to transfection with either c-Met 3′UTR, Notch-1 3′UTR, Notch-2 3′UTR or control reporter plasmids together with β-Gal plasmids for 48 hrs and 3′UTR reporter activity was measured by a luciferase assay and normalized to β-Gal. The results show that miR-34a expression downregulates c-Met, Notch-1 and Notch-2 luciferase activities in the cells. NICD = Notch intracellular domain. * = p<0.05.

Figure 2. Expression of miR-34a in human gliomas and correlation to c-Met and p53

A) miR-34a levels were measured by qRT-PCR in 11 glioblastoma surgical specimens and 6 normal brain samples and normalized to 18S rRNA measured in the same samples (arbitrary units). The results show that average levels of miR-34a in gliomas are lower than in normal brain. B) The p53 status of the glioma specimens described in (A) were determined. The blots show that average expression of miR-34a in wild-type p53 glioblastoma tumors (n=7) is significantly higher than miR-34a expression in mutant p53 tumors. C) c-Met expression in the same tissues described in (A) was measured by RT-PCR and normalized to β-Actin. Plotting of miR-34a vs. c-Met expressions shows an inverse correlation between them. D) Glioma cells were transfected with miR-34a for 48 hrs prior to measurement of c-Met mRNA levels by RT-PCR. The results show that miR-34a reduces c-Met mRNA levels. * = p<0.05.

Figure 3. miR-34a inhibits brain tumor cell proliferation, cell cycle progression, cell survival and cell invasion

Glioma and medulloblastoma cells were transfected with pre-miR-34a or pre-miR-con and subsequently assessed for cell proliferation by cell counting (A), for cell cycle by propidium iodide flow cytometry (B), for apoptosis by Annexin V flow cytometry and cleaved PARP immunoblotting (C) and for invasion by a transwell invasion assay (D). The results show that miR-34a strongly inhibits cell proliferation (A), induces cell cycle arrest (B), induces apoptosis (C) and inhibits transwell cell invasion (D). * = p<0.05.

Figure 4. c-Met and Notch expressions partially rescue cell cycle arrest and cell death induced by miR-34a

A) U87 cells were transfected with 5 μg pcDNA-Met or pcDNA control for 6 hrs prior to transfection with pre-miR-34a or pre-miR-con for 48 hrs. The cell cycle status of the transfected cells was analyzed by propidium iodide flow cytometry. c-Met expression changes were analyzed by immunoblotting. The results show that overexpression of 3′UTR-deleted c-Met partially rescues the cells from miR-34a-induced cell cycle arrest. B) 0308 stem cells were transfected with 2.5 μg pcDNA-Notch1 and 2.5 μg pcDNA-Notch2 or 5 μg pcDNA-control for 6 hrs prior to transfection with pre-miR-34a or pre-miR-con for 48 hrs. Apoptosis of the transfected cells was analyzed by Annexin V flow cytometry. Notch1 expression changes were analyzed by immunoblotting. The results show that overexpression of 3′UTR-deleted Notch1 and Notch2 partially rescues the cells from miR-34a-induced cell death. * = p<0.05.

Figure 5. miR-34a expression inhibits in vivo glioma xenograft growth

U87 cells were transfected with pre-miR-34a or pre-miR-con for 24 hrs. The transfected cells were implanted into the brains of immunodeficient mice (n=10). After 4 weeks, the mice were euthanized and the brains were cryosectioned and H&E stained. Tumor sizes were measured with computer-assisted image analysis. The results show that miR-34a inhibits in vivo glioblastoma xenograft growth. * = p<0.05.