PKM2 promotes glucose metabolism and cell growth in gliomas through a mechanism involving a let-7a/c-Myc/hnRNPA1 feedback loop - PubMed (original) (raw)

. 2015 May 30;6(15):13006-18.

doi: 10.18632/oncotarget.3514.

Yingyi Wang 1, Xincheng Chen 1, Yan Shi 1, Jiajia Wang 1, Junxia Zhang 1, Jin Qian 2, Ri Li 1, Tao Tao 1, Wenjin Wei 1, Qi Hu 1, Ning Liu 1, Yongping You 1

Affiliations

PKM2 promotes glucose metabolism and cell growth in gliomas through a mechanism involving a let-7a/c-Myc/hnRNPA1 feedback loop

Wenkang Luan et al. Oncotarget. 2015.

Abstract

Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than non-tumor cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We established a role for let-7a/c-Myc/hnRNPA1/PKM2 signaling in glioma cell glucose metabolism. PKM2 depletion via siRNA inhibits cell proliferation and aerobic glycolysis in glioma cells. C-Myc promotes up-regulation of hnRNPA1 expression, hnRNPA1 binding to PKM pre-mRNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let-7a, which functionally targets c-Myc, whereas hnRNPA1 blocks the biogenesis of let-7a to counteract its ability to downregulate the c-Myc/hnRNPA1/PKM2 signaling pathway. The down-regulation of c-Myc/ hnRNPA1/PKM2 by let-7a is verified using a glioma xenograft model. These results suggest that let-7a, c-Myc and hnRNPA1 from a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of glioma cells. These findings elucidate a new pathway mediating aerobic glycolysis in gliomas and provide an attractive potential target for therapeutic intervention.

Keywords: PKM2; aerobic glycolysis; c-Myc; glioma; glucose metabolism; hnRNPA1; let-7a microRNA.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1. PKM2 promotes glioma cells aerobic glycolysis and proliferation

(A) PKM2 expression profile in glioma cell lines, including U87, U251, H4 and T98G glioma cell lines. (B) Transfection efficiency of si-PKM2 was determined by Western blotting. (C) The proliferation ability of glioma cell lines U87 and U251 was measured by CCK8 assay. (D and E) The concentration of glucose and lactate in the culture medium was measured after the glioma cells were transfected with si-PKM2 or a negative control siRNA (NC). (F and G) ECAR and OCR were measured by the Glycolysis Stress and Cell Mito Stress tests in glioma cell lines after the cells were transfected with si-PKM2 or NC. *P < 0.05, **P < 0.01, ***P<0.001. Results are representative of at least three independent experiments.

Figure 2

Figure 2. Let-7a represses glioma cell glucose metabolism by inhibiting PKM2

(A) The effect of let-7a on the proliferation ability of U87 and U251 glioma cell lines was measured by CCK8 assay. (B and C) The concentration of glucose and lactate in the culture medium was measured after the glioma cells were transfected with let-7a or a negative control miRNA (NC). (D and E) ECAR and OCR were measured by the Glycolysis Stress and Cell Mito Stress tests in glioma cell lines after the cells were transfected with let-7a or NC. (F) Overexpression of let-7a decreased the expression of PKM2 in U87 and U251 cells as assessed by Western blotting. β-actin is shown as a loading control. *P < 0.05, **P < 0.01, ***P<0.001. Results are representative of at least three independent experiments.

Figure 3

Figure 3. HnRNPA1 is critical for the generation of PKM2 in glioma cells

(A) Schematic diagram of PKM splicing. hnRNPA1 promotes the formation of PKM2 (which includes exon 10) rather than the formation of PKM1 (which includes exon 9). [12] (B) Western blots identified hnRNPA1, PKM1 and PKM2 expression changes following transfection of U87 and U251 glioma cells with si-hnRNPA1. β-actin is shown as a loading control. (C) The expression of exon 9 and exon 10 were determined by qRT-PCR following transfection of si-hnRNPA1 or negative control siRNA (NC) into U87 and U251 cells. The expression of exon 9 and exon 10 was normalized to the expression of GAPDH mRNA. (D and E) The concentration of glucose and lactate in the culture medium was measured after the glioma cells were transfected with si-hnRNPA1 or NC. (F and G) ECAR and OCR were measured by the Glycolysis Stress and Cell Mito Stress tests in glioma cell lines after the cells were transfected with si-hnRNPA1 or NC. *P < 0.05, **P < 0.01, ***P<0.001. Results are representative of at least three independent experiments.

Figure 4

Figure 4. C-Myc regulates PKM2 via direct transcription of hnRNPA1 in glioma cells

(A) The hnRNPA1 promoter-luciferase reporter construct, pGL3-A1p, contains E boxes (CACGTG), which are located within a ~700nt hnRNPA1 promoter region and serve as putative c-Myc binding sites. The mutant construct, pGL3-A1pMut, has a mutated sequence within the E Box (CAGCTG). [12] (B) Over-expression of c-MYC in U87 and U251 glioma cells led to a marked increase in the luciferase activity of pGL3-A1p, without any change in the luciferase activity of pGL3-A1pMu. NC, negative control vector. (C) The expression of HnRNPA1 was detected using qRT-PCR following transfection with sh-Myc. (D) Western blots identified hnRNPA1 and PKM2 expression changes following transfection with sh-Myc alone or in combination with hnRNPA1. β-actin is shown as a loading control. (E and F) The concentration of glucose and lactate in the culture medium was measured after the glioma cells were transfected with sh-Myc alone or in combination with hnRNPA1. (G and H) ECAR and OCR were measured by the Glycolysis Stress and Cell Mito Stress tests in glioma cell lines. *P < 0.05, **P < 0.01, ***P<0.001. Results are representative of at least three independent experiments.

Figure 5

Figure 5. C-Myc is a functional target of let-7a that affects glucose metabolism in glioma cells

(A) Putative binding sites of Myc within the 3′UTR, as predicted by miRanda. (B) Over-expression of let-7a in U87 and U251 glioma cells led to a marked decrease in the luciferase activity of pGL3-WT-c-Myc-3′UTR-Luc, without any change in the luciferase activity of pGL3-mut–c-Myc-3′UTR-Luc. NC, negative control miRNA. (C) Western blots identified Myc, hnRNPA1 and PKM2 expression a change following transfection with let-7a alone or in combination with Myc. β-actin is shown as a loading control. (D and E) The concentration of glucose and lactate in the culture medium was measured after the glioma cells were transfected with let-7a alone or in combination with Myc. (F and G) ECAR and OCR were measured in glioma cell lines after transfection with let-7a alone or in combination with Myc. *P < 0.05, **P < 0.01, ***P<0.001. Results are representative of at least three independent experiments.

Figure 6

Figure 6. HnRNPA1 can act as a repressor of let-7a biogenesis in glioma cells

(A) Specific primers were designed to cover either the upstream region (Up.pri-let-7a-1) as a measure the transcription of the pri-miRNA; or to span the pri-let-7a-1 stem loop structure (pri-let-7a-1) as a measure of the levels of unprocessed pri-miRNA. [14]. (B) The expression of up. pri-let-7a-1, pri-let-7a-1 and mature let-7a were determined by qRT-PCR after transfection of si-hnRNPA1 or hnRNPA1 or a negative control vector (NC) into U87 and U251 cells. Expression was normalized to the expression of U6 rRNA. *P < 0.05, **P < 0.01. Results are representative of at least three independent experiments.

Figure 7

Figure 7. Let-7a inhibits glioma growth in vivo

The expression of let-7a, c-Myc, hnRNPA1 and PKM2 was examined by Immunohistochemical and In situ hybridization staining of sections from a glioma xenograft model in nude mice. The proteins were stained with ABC-peroxidase and diaminobenzene (brown color), while the cells were counterstained with hematoxylin (blue). Results are representative of at least three independent experiments.

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