MicroRNA-138 Increases Chemo-Sensitivity of Glioblastoma through Downregulation of Survivin

Abstract

Glioblastoma (GBM) is one of the most deadly cancers and poorly responses to chemotherapies, such as temozolomide (TMZ). Dysregulation of intrinsic signaling pathways in cancer cells are often resulted by dysregulated tumor suppressive microRNAs (miRNAs). Previously, we found miR-138 as one of tumor suppressive miRNAs that were significantly down-regulated in GBM. In this study, we demonstrated that ectopic over-expression of miR-138 sensitizes GBM cells to the treatment of TMZ and increased apoptotic cell death. Mechanistically, miR-138 directly repressed the expression of Survivin, an anti-apoptotic protein, to enhance caspase-induced apoptosis upon TMZ treatment. Using an intracranial GBM xenograft mice model, we also showed that combination of miR-138 with TMZ increases survival rates of the mice compared to the control mice treated with TMZ alone. This study provides strong preclinical evidence of the therapeutic benefit from restoration of miR-138 to sensitize the GBM tumor to conventional chemotherapy.

Keywords: BIRC5; glioblastoma (GBM); microRNA-138 (miR-138); survivin; temozolomide (TMZ).

Figure 1

Combination of TMZ with miR-138 over-expression increases inhibition of cell proliferation in vitro. Patient-derived primary GBM cells, (A) GBM12, (B) GBM28 and (C) GBM43, were transfected with miR-138 mimics or negative control (miR-Ctrl). After 4 days of transfection, viable cells were measured by CellTiter-Glo Luminescent Cell Viability Assay. All error bars indicates standard deviations (n = 3), and the p-values were determined by two-tailed student t-test. * p < 0.05, *** p < 0.001, n.s = not significant.

Figure 2

Over-expression of miR-138 combined with TMZ inhibits cell proliferation and increases apoptosis in vitro. (A) Cell proliferation analysis by fluorescence live cell imaging every four hours on GBM12-RFP cells after transfection of 25 nM miR-138 or miR-Ctrl. Left, the plot shows live cells imaged every 4 h to detect fluorescence intensity; Right, representative fluorescence images of GBM12-RFP cells at 96 h after the treatment of miRNA and TMZ. Scale bars indicate 100 µm. (B) Apoptosis analysis on GBM28 cells after transfection of 25 nM miR-138 or miR-Ctrl followed by Annexin V-PI double staining for flow cytometry. The scale bar indicates 100 µm. (C) Annexin V-positive cell population was considered to be apoptotic cells from cytograms. All error bars indicates standard deviations (n = 3), and the p-values were determined by two-tailed student t-test. *** p < 0.001.

Figure 3

The Cancer Genome Atlas Program (TCGA) database shows inverse correlation between BRIC5 and miR-138 in their expression. (A) BIRC5 expression is up-regulated, while (B) miR-138 expression is down-regulated in most types of glioma compared to that of normal tissues. (C) Direct comparison between BIRC5 and miR-138 clearly reveals inverse correlation in all types of glioma compared to normal tissues. All error bars indicates standard deviations, and Student t-test was used to determine the significance in difference between the two groups. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

miR-138 negatively regulates the expression of Survivin through direct targeting its 3′ UTR. (A) Western blotting on GBM cells (GBM12, GBM28 and GBM43) with transient overexpression of miR-138. The protein expression levels of Survivin were significantly decreased by imR-138 compared to miR-Ctrl treated GBM cells. Relative fold change of Survivin expression by miR-138 or miR-Ctrl was compared to the No miR control in each cell. (B) Schematic diagram of 3′ UTR sequences of Survivin containing a predicted miR-138 binding site (red letters). BIRC5-Mut construct contains the mutated sequences in the seed regions (blue letters). (C) Luciferase reporter assays for direct binding of miR-138 to the 3′ UTR of BIRC5. GBM cells were co-transfected with miR-138 and the luciferase reporter plasmid DNA containing BIRC5 3′ UTR, or BIRC5-Mut sequences respectively. The cells were further treated with TMZ for 4 days, and the repression of luciferase activity by miR-138 was analyzed by Dual Luciferase Assay kit. The relative luciferase activity values were normalized to Renilla luciferase activity as internal control. All error bars indicates standard deviations (n = 3), and the p-values were determined by two-tailed student t-test. * p < 0.05, *** p < 0.001, n.s = not significant.

Figure 5

Sensitization of GBM cells by miR-138 reduces tumorigenicity in orthotopic in vivo model. (A) Mouse survival Kaplan-Meier survival curve for mouse survival rates. Intracranial xenograft tumor was induced by implanting GBM cells transduced with miR-138 or miR-Ctrl expressing lentiviruses. (B) Mice brain tumor tissues were harvested subjected to western blotting to show the expression levels of Survivin. Expression of cleaved Caspase-3 and cleaved PARP was detected to show activation of apoptosis. Phosphorylated H2AX (p-H2AX) was used as an indicator for the DNA damage activity of TMZ. (C) Representative immunohistochemistry staining images of mice brain tumor tissue sections for the change of Survivin expression by miR-138. Ki76 was stained for cell proliferation. Scale bars indicate 100 µm.