MicroRNA-873 (miRNA-873) inhibits glioblastoma tumorigenesis and metastasis by suppressing the expression of IGF2BP1

Abstract

Glioblastoma multiforme (GBM) is known as a highly malignant brain tumor with a poor prognosis, despite intensive research and clinical efforts. In this study, we observed that microRNA-873 (miR-873) was expressed at low levels in GBM and that the overexpression of miR-873 dramatically reduced the cell proliferation, migration, and invasion of GBM cells. Our further investigations of the inhibition mechanism indicated that miR-873 negatively affected the carcinogenesis and metastasis of GBM by down-regulating the expression of IGF2BP1, which stabilizes the mRNA transcripts of its target genes. These results demonstrate that miR-873 may constitute a potential target for GBM therapy.

Keywords: GBM; Gene Expression; Glioblastoma; IGF2BP1; Insulin-like Growth Factor (IGF); MicroRNA (miRNA); MicroRNA Mechanism; MicroRNA miR-873.

FIGURE 1.

Analysis of miR-873 in GBM tissues and adjacent normal tissues. A, the relative expression level of mature miR-873 in six pairs of tumor issues and adjacent normal counterparts using real-time RT-PCR. The lines represent the means of independent experiments in each group. * indicates p < 0.005. B, the relative levels of miR-873 were measured in four normal human astrocytes and six GBM cell lines, A172, T98G, U87, U373, U251, and U138. Error bars represent S.E. * indicates p < 0.05 versus normal. C and D, miR-873 expression detected by primer extension. T98G, A172, U373, and U87 are glioblastoma cell lines. 5 mg of total RNA were used for each reaction. Free probe is shown as a negative control. Primer extension reactions were resolved on 15% Tris-borate-EDTA-urea polyacrylamide gels and exposed to film for 50 min.

FIGURE 2.

The increased level of miR-873 inhibits the proliferation but induces the apoptosis of glioma cells in vitro. A, the relative levels of miR-873 in two GBM cell lines, U87 and U373, were determined after the cells were transfected with miR-873 mimics or miR-control mimics using real-time RT-PCR and primer extension assay. B, the relative cell viability in U87 cells was measured after the cells were transfected with miR-873 mimics or miR-control mimics using MTT assays. C, the relative cell viability of U373 cells was detected using MTT assays after the cells were transfected with miR-873 mimics or miR-control mimics. D, the colony formation of miR-873-U87 cells and miR-873-U373 cells was quantified and compared with that of the GBM cells transfected with miR-control mimics in their respective groups. E, the representative micrographs of the colony formation assays are shown. F, the cell cycle distributions of the U87 cells transfected with miR-873 mimics or miR-control mimics were detected and compared. G, the cell cycle distributions of the U373 cells transfected with miR-873 mimics or miR-control mimics were detected and compared with each other. H, the apoptosis rate of the two GBM cell lines transfected with miR-873 mimics were measured and compared with that of the cells transfected with miR-control mimics. Each bar or point represents the mean of three independent experiments. I, the U87 GBM tumor growth in vivo was determined based on the tumor volume, which was calculated based on weekly measurements after injection. J, a representative image of in vivo tumor growth is shown. In this panel, the weights of the tumors generated from the GBM cells transfected with miR-873 mimic in three nude mice were measured and compared with the control groups. Error bars represent S.E. * means p < 0.05.

FIGURE 3.

The transfection of GBM cells with miR-544 inhibits the migration and invasion of the cells in vitro. A, the migration abilities of miR-873-U87 cells and miR-873-U373 cells were quantified and compared with that of the cells transfected with miR-control mimics in their respective groups. B, the representative micrographs of the migration of miR-873-U87 cells and miR-873-U373 cells are shown. C, the invasiveness of miR-873-U87 cells and miR-873-U373 cells was quantified and compared with that of cells transfected with miR-control mimics in their respective groups. D, the representative micrographs of the invasion of miR-873-U87 and miR-873-U373 cells are shown. E, the wound-healing assay showed that the migration of miR-873-U87 cells differed from that of cells transfected with miR-control mimics at different time points. F, the wound-healing assay showed that the migration of miR-873-U373 cells differed from that of cells transfected with miR-control mimics at different time points. The motility is expressed as the percentage of migration at the zero time point. Each bar represents the mean of three independent experiments. Error bars represent S.E. * indicates p < 0.05.

FIGURE 4.

miR-873 directly targeted the IGF2BP1 gene. A, the potential targeted genes are listed based on the predictions by the four algorithms. The gene to be further analyzed, IGF2BP1, was within the shaded area. The structures of the RNA duplexes formed by miR-873 and its putative binding sequences with the predicted mfe were located on the right. B, the 3′-UTR fragments of IGF2BP1 gene and its mutant are indicated in boxes. The putative base pair region is labeled with vertical lines between the words representing the bases. C, the relative firefly activities generated by the luciferase reporter plasmids containing wild-type or mutant IGF2BP1 3′-UTR in GBM cells with miR-873 or control mimics were determined using the Dual-Luciferase assays. D, Western blot analyses of the IGF2BP1 protein level were performed following treatment of U87 cells and U373 cells with miR-873, anti-miR-873, or controls. E, quantitative RT-PCR analyses of the IGF2BP1 mRNA level were performed following the treatment of U87 cells and U373 cells with miR-873 or control mimics. GAPDH was used as a control. Error bars indicate S.D. *, p < 0.05.

FIGURE 5.

The inhibition of cell growth and invasion caused by miR-873 was reversed by the overexpression of IGF2BP1 in GBM cells. A, the relative expression levels of IGF2BP1 were detected in the GBM cells transfected with miR-873 or control mimics and in cells co-transfected with miR-873 and pcMV6.0/IGF2BP1 using quantitative RT-PCR. B, the relative protein levels of IGF2BP1 were determined using Western blot in the GBM cells transfected with miR-873 or control mimics and in cells co-transfected with miR-873 and pcMV6/IGF2BP1. C and D, the ability of GBM cell transfected with miR-873 or control mimics and co-transfected with miR-873 and pcMV6/IGF2BP1, measured by MTT or colony formation assays. E, the representative colony images were shown. F, Apoptosis assay was performed in each group of transfected GBM cells. G and H, the migration and invasion abilities of the GBM cells transfected with miR-873 or control mimics and cells co-transfected with miR-873 and pcMV6/IGF2BP1 are presented using Transwell assays. The experiments were performed in triplicate. Error bars indicate S.D. *, p < 0.05.

FIGURE 6.

The up-regulation of miR-873 suppresses the expression of MKI67, C-MYC, PTEN, and CD44 in GBM cells. A, the levels of MKI67 and c-MYC mRNA in U87 cells after transfection with miR-873 mimics or control mimics were analyzed by real-time RT-PCR. B, the levels of PTEN and CD44 mRNA in U87 cells transfected with miR-873 mimics or control mimics were analyzed by real-time RT-PCR. C, the levels of MKI67 and c-MYC mRNA in U373 cells transfected with miR-873 mimics or control mimics were analyzed by real-time RT-PCR. D, the levels of PTEN and CD44 mRNA in U373 cells transfected with miR-873 mimics or control mimics were analyzed by real-time RT-PCR. E, the protein levels of MKI67 and c-MYC in U87 and U373 cells transfected with miR-873 mimics were determined relative to the corresponding cells transfected with control mimics by Western blotting. F, the protein levels of PTEN and CD44 in U87 and U373 cells transfected with miR-873 mimics were determined relative to the corresponding cells transfected with control mimics by Western blotting. GAPDH mRNA served as the control to determine the mRNA abundance relative to the controls, and GAPDH protein served as the control to determine the protein abundance. The statistical significance was determined with Student's t test: *, p < 0.005. Error bars indicate the S.D. of at least three independent analyses.

FIGURE 7.

The up-regulation of IGF2BP1 expression caused by the decreased level of miR-873 expression is involved in the progression of GBM. A, analysis of IGF2BP1 in GBM tissues and adjacent normal tissues. B, analysis of IGF2BP1 in GBM cell lines and normal cells. C, the inverse relationship between the expression of IGF2BP1 and miR-873. D, GBM and normal ones tissues were stained with specific antibodies to IGF2BP1. Representative micrographs were framed and enlarged. E, the suggested molecular mechanism of the tumorigenesis and metastasis of GBM. Error bars indicate the S.E. of at least three independent analyses.