Effect of glycolysis inhibition by miR-448 on glioma radiosensitivity

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OBJECTIVE

Although glucose metabolism reengineering is a typical feature of various tumors, including glioma, key regulators of glycolytic reprogramming are still poorly understood. The authors sought to investigate whether glycolysis inhibition by microRNA (miR)–448 increases radiosensitivity in glioma cells.

METHODS

The authors used glioma tissue samples from glioma patients, cells from glioblastoma (GBM) cell lines and normal human astrocyte cells, and subcutaneous tumor–bearing U87 cells in mice to examine the effects of signaling regulation by miR-448 in the response of glioma tissues and cells to radiation treatment. Techniques used for investigation included bioinformatics analyses, biochemical assays, luciferase reporter assays, and establishment of subcutaneous tumors in a mouse model. Glucose consumption, LDH activity, and cellular ATP were measured to determine the ability of glioma cells to perform glycolysis. Expression of HIF-1α was measured as a potential target gene of miR-448 in glycolysis.

RESULTS

miR-448 was detected and determined to be significantly downregulated in both glioma tissues from glioma patients and GBM cell lines. Furthermore, miR-448 acted as a tumor-inhibiting factor and suppressed glycolysis in glioma by negatively regulating the activity of HIF-1α signaling and then interfering with its downstream regulators relative to glycolysis, HK1, HK2, and LDHA. Interestingly, overexpression of miR-448 increased the x-radiation sensitivity of glioma cells. Finally, in in vivo experiments, subcutaneous tumor–bearing U87 cells in a mouse model verified that high expression of miR-448 also enhanced glioma radiosensitivity via inhibiting glycolytic factors.

CONCLUSIONS

miR-448 can promote radiosensitivity by inhibiting HIF-1α signaling and then negatively controlling the glycolysis process in glioma. A newly identified miR-448–HIF-1α axis acts as a potentially valuable therapeutic target that may be useful in overcoming radioresistance in glioma treatment.

ABBREVIATIONS FACS = fluorescence-activated cell sorting; GBM = glioblastoma; KPS = Karnofsky Performance Status; miR = microRNA; miRNA = microRNA; miR-NC = miR-448 negative controls; NHA = normal human astrocyte; NSCLC = non–small cell lung cancer; PCR = polymerase chain reaction; qRT-PCR = quantitative RT-PCR; RT-PCR = real-time PCR; UTR = untranslated region.

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Article Information

Correspondence Xiao Yue: The Affiliated Hospital of Xiangnan University, Chenzhou, Hunan, People’s Republic of China. yuexiaolfm@163.com.

INCLUDE WHEN CITING Published online April 19, 2019; DOI: 10.3171/2018.12.JNS181798.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

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Figures

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    miR-448 downregulation in glioma tissues confers poor prognosis. A: miR-448 analysis by qRT-PCR in glioma and normal tissue samples from study patients shows a significant difference in the expression of miR-448 between glioma tissues and nonneoplastic brain tissues. B: miR-448 analyzed by qRT-PCR in tissue samples from patients with low- and high-grade glioma shows increased levels of miR-448 expression in low-grade glioma (LGG) versus high-grade glioma (HGG). C: miR-448 expression was significantly decreased in glioma cell lines (U87, U251, A172, and LN229) compared with NHA cells. D: Kaplan-Meier survival curve analysis of glioma patients with high and low expression of miR-448. Cumulative (Cum) survival rates in patients with lower expression of miR-448 were significantly shorter (p < 0.01) than rates in patients with higher expression of miR-448. **p < 0.01.

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    miR-448 suppresses the proliferation, migration, and invasion of GBM cells in vitro and in vivo. A: Growth curves from U87 (left) and LN229 (right) GBM cells transfected with miR-448 mimics or miR-NC control assessed by CCK8 assay show significant interference of miR-448 on proliferation of GBM cells. B: For assessment of the colony formation efficiencies of the transfected cells, the number of cell colonies on soft agar was determined and was found to be significantly decreased for the miR-448 mimic groups compared with the miR-NC controls. C: miR-448–induced apoptosis detected by FACS analysis showed that the percentage of apoptotic cells was significantly increased in the miR-448 groups. D and E: miR-448 significantly reduced the migration and invasive capacity of GBM cells, as evaluated by transwell (D) and wound healing assays (E), respectively. F: U87 xenografts in mice treated with miR-448 showed significant growth inhibition compared with controls. G: Expression of miR-448 was detected by use of qRT-PCR in tumor xenografts in the mouse model. **p < 0.01.

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    miR-448 directly targets the 3′UTR of HIF-1α and represses its expression. A: HIF-1α was analyzed in glioma and normal tissue samples from study patients and was found to be inhibited in glioma patients with high miR-448 levels and enhanced in patients with low levels. **p < 0.01. IHC = immunohistochemistry. B: Inverse correlation of miR-448 with HIF-1α. C: As indicated by the sequence alignment between miR-448 and the 3′UTR of HIF-1α, the 3′UTR of HIF-1α contains highly conserved targeting sites of miR-448. D: Western blot analysis was performed to evaluate the expression level of HIF-1α in U87 and LN229 GBM cells transfected with miR-448 mimics and shows that miR-448 overexpression decreased the level of HIF-1α. E: pGL3-WT–HIF-1α–3′UTR-Luc and pGL3-MUT–HIF-1α–3′UTR-Luc reporters were transfected with miR-448 mimics in U87 and LN229 GBM cells. Luciferase activity of the reporter plasmid was significantly suppressed in wild-type (Wt) HIF-1α–3′UTR compared with the HIF-1α–3′UTR mutant (Mut) group when transfected with miR-448. **p < 0.01.

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    miR-448 inhibits glycolysis mediated by HIF-1α in GBM cells. A: Western blot analysis of HIF-1α, HK1, HK2, and LDHA protein expression showed that miR-448 overexpression reduced levels of HIF-1α and downstream regulators in U87 and LN229 GBM cells. B–E: Glucose consumption was measured by microplate assay (B) to evaluate glucose metabolism. Lactic acid production in glioma cells (C) was inhibited by miR-448 overexpression. Activity of LDHA, analyzed by gel activity assay (D), and ATP production, measured using a luciferin/luciferase assay (E), were also inhibited by miR-448 overexpression in GBM cells. Nontreated cells were used for normalization. The value for nontreated cells was set as 1. **p < 0.01.

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    miR-448 enhances radiosensitivity of GBM cells to x-radiation. A: Cell viability is expressed as the percentage relative to scrambled control cells. miR-448 significantly suppressed cell viability compared to control. B: Clonogenic assays of cells treated with 0-, 2-, 4-, or 6-Gy radiation doses. Cellular sensitivity to radiation was significantly increased when cells were transfected with miR-448 mimics. C: Flow cytometry analysis of apoptosis using annexin V and propidium iodide in cells treated with a 0- or 10-Gy radiation dose. miR-448 mimics significantly augmented the percentage of apoptotic cells induced by 10-Gy irradiation. D: In subcutaneous xenograft tumors in the mouse model, the tumor volume of each group was detected. Radiation alone significantly reduced tumor growth, and combining radiation with miR-448 enhanced the effect of radiation in vivo. E: Expression of HIF-1α downstream genes was detected by Western blot. Overexpression of miR-448 significantly downregulated the level of HIF-1α, HK1, HK2, and LDHA proteins in the treated xenograft tumors. **p < 0.01.

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