MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells
- First Online:
- Cite this article as:
- Tanaka, H., Sasayama, T., Tanaka, K. et al. J Neurooncol (2013) 111: 273. doi:10.1007/s11060-012-1027-9
- 879 Views
MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression and contribute to cell proliferation, differentiation and metabolism. Our previous study revealed the extensive modulation of a set of miRs in malignant glioma. In that study, miR microarray analysis demonstrated the upregulation of microRNA-183 (miR-183) in glioblastomas. Therefore, we examined the expression levels of miR-183 in various types of gliomas and the association of miR-183 with isocitrate dehydrogenase 2 (IDH2), which has complementary sequences to miR-183 in its 3′-untranslated region (3′UTR). In present study, we used real-time PCR analysis to demonstrate that miR-183 is upregulated in the majority of high-grade gliomas and glioma cell lines compared with peripheral, non-tumorous brain tissue. The mRNA and protein expression levels of IDH2 are downregulated via the overexpression of miR-183 mimic RNA in glioma cells. Additionally, IDH2 mRNA expression is upregulated in glioma cells expressing anti-miR-183. We verified that miR-183 directly affects IDH2 mRNA levels in glioma cells using luciferase assays. In malignant glioma specimens, the expression levels of IDH2 were lower in tumors than in the peripheral, non-tumorous brain tissues. HIF-1α levels were upregulated in glioma cells following transfection with miR-183 mimic RNA or IDH2 siRNA. Moreover, vascular endothelial growth factor and glucose transporter 1, which are downstream molecules of HIF-1α, were upregulated in cells transfected with miR-183 mimic RNA. These results suggest that miR-183 upregulation in malignant gliomas induces HIF-1α expression by targeting IDH2 and may play a role in glioma biology.
MicroRNAs (miRs) are small, non-coding RNAs transcribed from DNA that are 18–24 nucleotides in length and subsequently regulate gene expression and contribute to cell proliferation, apoptosis, differentiation and metabolism [1, 2]. miRs are processed from primary transcripts, known as primary miRs, into short stem–loop structures called premature miRs and then finally processed into mature miRs . A single miR has the capacity to regulate a large number of target messenger RNAs (mRNAs), and the main function of miRs is to downregulate gene expression [4, 5]. It has been proposed that depending on the role of their mRNA targets, miRs can function either as tumor suppressors or as oncogenes . In glioblastoma, which is one of the most malignant brain tumors, a number of miRs are reported to display aberrant expression patterns [7–11]. Using miR microarray analysis, we previously described that miR-10b, miR-21, miR-183, miR-92b, and miR-106b are highly expressed in glioblastomas compared with normal brain tissue . Several other reports have also identified these miRs as being upregulated in glioblastomas [13–16]. Recently, the expression of miR-183 was identified as being upregulated in colorectal cancer and hepatocellular carcinoma and was also reported to be involved in tumor invasion or migration [17–20].
Isocitrate dehydrogenases (IDHs) are a group of enzymes involved in monocarbon metabolism, which catalyze the conversion of isocitrate to α-ketoglutarate by oxidative decarboxylation [21–25]. IDH1 is a cytosolic and peroxisomal enzyme, whereas IDH2 and IDH3 are mitochondrial enzymes [25, 26]. Only IDH1 and IDH2 are known to be mutational targets in human cancers. Recently, mutations in IDH1 and IDH2 were reported in association with gliomas, acute myeloid leukemia and other cancers [27–32].
In this study, we determined that miR-183 is upregulated in a majority of malignant (high-grade) gliomas compared with peripheral, non-tumorous brain tissue. Because miR-183 has complementary sequences in the 3′-untranslated region (3′UTR) of IDH2 mRNA, we hypothesized that miR-183 regulates IDH2 expression. We demonstrated that miR-183 potently inhibits IDH2 expression at both the mRNA and protein levels in glioma cell lines. We next demonstrated that the expression of IDH2 mRNA and protein was downregulated in most malignant gliomas compared with peripheral, non-tumorous brain tissue. Additionally, we demonstrated that the overexpression of miR-183 or knockdown of IDH2 by siRNA in glioma cells elevated the protein levels of HIF-1α. These results imply that overexpressing miR-183 might repress the mitochondrial enzyme IDH2 and upregulate HIF-1α in glioma cells.
Materials and methods
Five GBM cell lines (U251, U87MG, T98G, A172 and SF126) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai tesque, Kyoto, Japan) containing glutamine, 10 % fetal bovine serum, and penicillin/streptomycin. Immortalized human astrocyte cell line (SVGp12) were maintained in Eagle’s minimum essential medium (E-MEM, Wako, Osaka, Japan) containing glutamine, 10 % fetal bovine serum, and penicillin/streptomycin. Cells were grown in a 37 °C incubator with 5 % CO2.
Glioma tissues were obtained from therapeutic procedures performed as part of routine clinical management at the Department of Neurosurgery at the University of Kobe. Tissue samples were resected during surgery and immediately frozen in liquid nitrogen for subsequent investigation. A total of 88 glioma specimens (53 glioblastomas, 14 anaplastic astrocytomas, 3 anaplastic oligodendrogliomas, 10 diffuse astrocytomas, 5 oligodendrogliomas, and 3 pilocytic astrocytomas) and 6 peripheral brain tissue samples were included in this study. This study was approved by the ethics committee at the Kobe University Graduate School of Medicine.
The miR-183 mimic RNA (MISSION microRNA Mimic-has-miR-183) and the negative control RNA (MISSION microRNA Mimic, Human, Negative Control 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-miR-183 (mirVana™ Inhibitors) and negative control (mirVana™ Inhibitors Negative Control-1) were purchased from Applied Biosystems/Ambion (Foster City, CA, USA). The IDH2 siRNA and negative control siRNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany) was used to transfect the miR-183 mimic/inhibitor or IDH2 siRNA into cells, and the cells were then subjected to real-time PCR and western blotting, according to the manufacturer’s instructions. The miR-183 mimic/inhibitor or IDH2 siRNA were transfected at a final concentration of 20 nM. A total of 1.0 × 105 glioma cells were plated into a 12-well plate in 1 ml of DMEM containing serum. The miR-183 mimic/inhibitor or siRNA was diluted in 100 μl OptiMEM (Invitrogen, Carlsbad, CA, USA) without serum. Then, 6 μl of HiPerFect Transfection Reagent was added to the diluted miR-183 mimic/inhibitor or siRNA solution and mixed, and the complexes were added drop-wise onto the cells. The cells were incubated with the transfection complexes under their normal growth conditions (37 and 5 % CO2) for 48–96 h (mRNA assay: 48–72 h, protein assay: 72–96 h) before the assays were performed. A Cy3 dye-labeled Anti-miR Negative Control (Applied Biosystems/Ambion) was used to monitor the transfection efficiency of the transfection experiment. Transfection efficiency was greater than 80 % in all glioma cell lines (U251, U87, A172, T98, and SF126) (Supplementary Fig. 1B).
We used three sets of luciferase reporter systems (pLightSwitch-3′UTR-empty-Luc vector and pLightSwitch-wild-type-IDH2-3′UTR-Luc vector, pLightSwitch-mutant-IDH2-3′UTR-Luc vector) (Switchgear genomics, Menlo Park, CA, USA) (Fig. 3a). The pLightSwitch-mutant-IDH2-3′UTR-Luc vector was generated from pLightSwitch-wild-type-IDH2-3′UTR-Luc vector by replace the binding site of miR-183 using QuikChange Site-Directed Mutagenesis Kit (Agilent technologies, Santa Clara, CA, USA). The empty vector contains neither the 3′UTR nor a control insert. DharmaFECT Transfection Reagent (Promega, Fitchburg, Wisconsin, USA) was used to co-transfect the miR and plasmid into the cells according to the manufacturer’s protocol. After transfection, the cells were incubated in 96-well plates and harvested for the luciferase assays approximately 48 h later. The Dual-Luciferase Reporter Assay System (Promega) was used to measure the luciferase activity in the cells transfected with either the pLightSwitch-3′UTR-control or the pLightSwitch-3′UTR-IDH2 vector and pGL4-renilla luciferase-control vector using a GloMax® 96 Microplate Luminometer (Promega). Expression was calculated as the relative firefly luciferase activity normalized to the activity of the co-transfected control renilla luciferase.
Western blotting and antibodies
Cells were lysed on ice for 30 min in lysis buffer containing 1.0 % NP-40, 0.1 % SDS, 100 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM ZnCl2, 50 mM NaMoO4, 10 mM NaF, 0.1 mM sodium vanadate, and a protease inhibitor cocktail. Electrophoresis was performed for approximately 90 min at 20 mA in a 1.0 mm polyacrylamide gel. The proteins were transferred electrophoretically at 120 mA for 60 min and assessed by immunoblotting using primary antibodies against IDH2 (Abcam, Cambridge, MA, USA), HIF-1α (H1alpha67) (Novus Biologicals, Littleton, CO, USA) and ß-actin (Ambion, Inc., Austin, TX, USA). Immunoblot development was performed as recommended by the protocol provided in the ECL Advance Western Blotting detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). For quantitative analysis by normalizing to β-actin, Image J software (National Institutes of Health (NIH), Bethesda, MD, USA) was used.
Real-time RT-PCR for miRs
Reverse transcription was performed using the TaqMan® microRNA Reverse Transcription Kit, according to the manufacturer’s instructions. TaqMan® microRNA assays (Applied Biosystems) were used to quantify mature miR expression, as previously described . RNU6 (Applied Biosystems) was used as the endogenous control for the miR expression studies. Real-time PCR reactions for miRs were performed in triplicate in 20 μl volumes. Quantitative miR expression data were acquired and analyzed using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems) and the ΔΔ-Ct method.
Real-time RT-PCR for IDH2, HIF-1α, VEGF, and GLUT1
Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions. Expression levels of IDH2 and HIF-1α mRNA were quantitatively assayed by real-time RT-PCR using TaqMan® Gene Expression Assays (Applied Biosystems). The 18S ribosomal RNA transcript was used as the endogenous control. Quantitative mRNA expression data were acquired and analyzed by the ΔΔ-Ct method using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems).
Immunocytochemistry of IDH2
Cells were fixed with 4 % paraformaldehyde in sodium phosphate (NaPi) buffer (pH 7.4) for 15 min and then incubated in 0.3 % triton-X/PBS for 15 min. Both endogenous peroxidase and biotin were blocked, and the cells were incubated with antibodies against IDH2 (Abcam) at 4 °C overnight. The cells were allowed to react with peroxidase-conjugated anti-mouse IgG monoclonal antibody (Histofine Simple Stain MAX-PO; Nichirei, Tokyo, Japan) for 60 min and visualized by immersing the sections in 0.03 % diaminobenzidine solution containing 2 mM hydrogen peroxide for 5 min. The nuclei were lightly stained with Mayer’s hematoxylin.
α-Ketoglutarate (α-KG) Assay
α-KG Assay was performed using an α-KG Assay Kit (Abcam), according to the manufacturer’s instructions. Cells were rapidly homogenized with 100 μl of ice cold buffer containing 1 % NP-40, and 1–50 μl of supernatant was transferred into triplicate wells of a 96-well plate. For each well, a total 50 μl Reaction Mix containing α-KG Assay Buffer, α-KG Converting Enzyme, α-KG Enzyme Mix, and α-KG Probe were prepared and 50 μl of the Reaction Mix were added to each well. Samples were incubated for 30 min at 37 °C, and then measured OD at 570 nm. The background control was subtracted from the α-KG reading.
The Mann–Whitney U-test or Student’s t test was used to analyze the differences between groups, and a p value of less than 0.05 was considered to be significant. Statistical analyses were performed using the SPSS 12.0 software package (SPSS Inc., Chicago, IL, USA).
miR-183 is upregulated in gliomas
miR-183 downregulates the expression of IDH2
miR-183 directly targets IDH2
mRNA and protein expression levels of IDH2 are downregulated in malignant gliomas
miR-183 overexpression elevates the expression of HIF-1α
As noted above, we found that miR-183 is upregulated in a majority of malignant gliomas. Additionally, the overexpression of miR-183 inhibits the expression of IDH2, which is one of the mitochondrial enzymes in glioma cells related to the TCA cycle. Additionally, we determined that IDH2 expression was downregulated in a majority of malignant gliomas. Moreover, we demonstrated that the overexpression of miR-183 increased both the protein and mRNA levels of HIF-1α in glioma cells. The aberrant expression of miR-183 in various cancers, such as lung cancer, colon cancer, hepatocarcinoma, and medulloblastoma have been reported, suggesting that there is a close correlation between miR-183 and cancer development [34–36]. It is reported that miR-183 regulates the expression of ezrin, integrin β1, and kinesin 2α and affects tumor cell invasion and migration [19, 37]. Additionally, miR-183 inhibits TGF-β-1-induced apoptosis via the downregulation of PDCD4 expression in human hepatocellular carcinoma cells . Our study demonstrates that miR-183 upregulated HIF-1α in glioma cells, which may in turn contribute to glioma formation or survival.
Interestingly, we identified a direct relationship between miR-183 and IDH2 in glioma cells. To our knowledge, this is the first report demonstrating the interaction between miR-183 and IDH2 in glioma cell lines. Although the expression of miR-183 in U87 and U251 is significantly lower than in A172 and SF126, miR-183 mimic RNA has very similar effects on IDH2 expression. This may be because the expression levels of IDH2 mRNA are different in each cell line. Moreover, because the miR-183 mimic RNA also reduced the level of IDH2 mRNA in human astrocyte cell line SVGp12, IDH2 inhibition by miR-183 is not a tumor cell specific phenomenon.
Recent studies have reported a high frequency of IDH1 mutations in gliomas. However, compared to IDH1, IDH2 mutations are much less common in gliomas; however, IDH2 mutations are more common than IDH1 mutations in acute myeloid leukemia . Although IDH2 mutations are rare in gliomas, our analysis demonstrated that reduced IDH2 expression is frequently observed in malignant glioma. Therefore, we considered that the increased expression of miR-183 reduces the level of IDH2 in glioma cells. Very recently, Vohwinkel et al.  reported that elevated CO2 concentrations (hypercapnia) lead to increased levels of miR-183, which downregulates IDH2 and impairs mitochondrial function and cell proliferation in fibroblasts and alveolar epithelial cells.
IDH has the ability to convert isocitrate to α-KG. The present study revealed that the downregulation of IDH2 by miR-183 reduces the cellular levels of α-KG in glioma cells (Fig. 5a). Prolyl hydroxylases (PHDs), which use α-KG as substrates, hydroxylate HIF-1α to promote its proteasomal degradation in the presence of oxygen [40, 41]. Zhao et al.  previously reported the association between cytoplasmic IDH1 and HIF-1α . The authors revealed that mutant IDH1 dominantly inhibited wild-type IDH1 resulting in a decrease in cellular levels of α-KG, which causes an elevation in HIF-1α protein levels. Based on this mechanism, we assumed that a miR-183-mediated reduction in IDH2 might elevate the expression of HIF-1α protein. In addition, our data revealed that miR-183 elevates not only HIF-1α protein but also HIF-1α mRNA levels; however, the mechanism of this is unclear. To elucidate this phenomenon, further study is necessary.
Although the mechanism for how miR-183 affects HIF-1α is not completely understood, we provide evidence that the overexpression of miR-183 increases HIF-1α expression. HIF-1α activates critical pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) and erythropoietin. Furthermore, HIF-1α stimulates glucose uptake by regulating the expression of the glucose transporters GLUT1 and GLUT3 and regulates a number of glycolysis-related genes . Moreover, HIF-1α also plays a key role in suppressing mitochondrial activity by inducing pyruvate dehydrogenase-kinase1 (PDK1), which inactivates pyruvate dehydrogenase (PDH) . PDH provides the link between glycolysis and the TCA cycle by catalyzing the conversion of pyruvate into acetyl-CoA. As a result, the inactivation of PDH leads to suppression of both the TCA cycle and mitochondrial respiration.
Tumor cells obtain energy by using aerobic glycolysis, and they exhibit a concomitant defect in mitochondrial respiration. In addition, tumor cells require ongoing lipid synthesis. Fatty acids are essential components of all biological lipid membranes and are critical substrates for energy metabolism. Under aerobic conditions in tumor cells, cell metabolism shifts from an oxidative to a glycolytic mode, which leads to an increase in the mitochondrial concentration of citrate . Citrate is an essential component for fatty acid synthesis [45, 46]. Therefore, we speculate that the downregulation of IDH2 by miR-183 overexpression might induce mitochondrial dysfunction and de novo fatty acid synthesis.
In conclusion, the major findings presented in this study are that miR-183 directly targets the 3′UTR of IDH2 mRNA in glioma cells, which induces the downregulation of IDH2 and the upregulation of HIF-1α. Because HIF-1α plays a significant role in angiogenesis, metabolism and survival in tumor cells, the overexpression of miR-183 might contribute to gliomagenesis. Further studies are necessary to clarify the association between miR-183 and tumor formation, growth, and metabolism, and the mechanisms responsible for the upregulation of HIF-1α by miR-183.
We thank Mitsuharu Endo and Ryosuke Doi (Department of Physiology and Cell Biology, Kobe University, Japan) for helping with the luciferase reporter assays. We also thank Mariko Ueda for helping with the immunocytochemical analysis. This work was supported in part by a Grant-in-Aid for Scientific Research to Takashi Sasayama (22591610) and Katsu Mizukawa (22791344) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Conflicts of interest