PKM2 Contributes Glucose Metabolism And Cell Growth In Glioma By Involving Let-7a/c-Myc/HnRNPA1 Loop

Research background:The change of energy metabolism is listed as one of the ten hallmarks of Cancer. The study of tumor cell metabolism has become the hot spot in tumor research. Normal differentiated cells extract energy from glucose chiefly through oxidative phosphorylation in aerobic condition; metabolize glucose to lactate through glycolysis in hypoxic conditions. However, the tumor cells metabolize more glucose to lactate in two different conditions. The phenomenon is termed as the Warburg effect. Pyruvate kinase (PK) is the last and the most important pacemaker enzyme of glycolysis. PKM1 and PKM2 are two isoforms of the PK. The PKM1 promotes oxidative phosphorylation, whereas PKM2 primarily promotes aerobic glycolysis. he PKM2 and PKM1 arise from alternative splicing of transcripts of the PKM gene. The PKM2 contain exclusive exon 10, the PKM1 is exon 9. Three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins hnRNPAl, hnRNPA2 and polypyrimidine tract binding protein (hnRNPI, PTB), bind repressively to exon 9. They are critical for the generation of PKM2 in glioma. Meanwhile, the oncogenic transcription factor c-Myc up-regulate the transcription of hnRNPAl, hnRNPA2 and hnRNPI. MicroRNA are 19 to 25 nucleotide non-protein-coding small RNA, it can silence mRNA translation through binding to specific sites on mRNA. It is involved in tumor cells proliferation, invasion, apoptosis and other important processes. Meanwhile, miRNA may play an important role on cancer cell metabolism. Let-7a as a tumor suppressor function of miRNA. Many reports determined that let-7a down-regulate the expression of c-Myc in many human malignancies. In addition, HnRNPA1 could hinder the expression of let-7a. In this study, we investigated the role of let-7a in the glucose metabolism of glioma cell for the first time. The feedback loop of let-7a, c-Myc and hnRNPAl were confirmed in glioma cells. This loop could influence the growth and glucose metabolism of glioma cells by regulates the expression of PKM2 protein. In addition, we confirmed let-7a/c-Myc/hnRNPA1/pkm2 signaling in vivo through using nude mouse glioma xenograft. Taken together, these results provide theoretical basis for the let-7a as glioma diagnostic and therapeutic targets.Methods:1. The expression of PKM2 were down-regulated by si-PKM2 in human glioma cell line U87 and U251 cells. CCK8 assay, cell flow cytometry were used to access the proliferation ability and cell cycle in glioma cells. The concentration of glucose and lactate in the cell culture medium. ECAR and OCR were measured.2. The effects of the proliferation and the levels of glucose metabolism were evaluated by CCK8 assay, Glucose and Lactate Assay Kit, ECAR and OCR in U87 and U251 cells after transfected with let-7a. Expression of PKM2was tested with western blot3. Glioma cell line U87 and were transfected with si-HnRNPA1, western blot assay was used to test expression of HnRNPA1、PKM1 and PKM2, exon 9 and exon10 was assessed by quantitative RT-PCR. The concentration of glucose and lactate in U87 and U251 cell culture medium, ECAR and OCR were measured.4. Luciferase reporter assay and western blot assay were used to analysis whether HnRNPA1 is the target of c-Myc. The concentration of glucose and lactate, ECAR and OCR were measured in U87 and U251 cells after transfected with sh-Myc or co-transfected with sh-Myc and hnRNPAl.5. Bioinformatics, Luciferase reporter assay and western blot assay were used to analysis whether c-Myc is the target of let-7a. Glucose and Lactate Assay Kit, ECAR and OCR were used to evaluate the level of glucose metabolism after transfected with let-7a or co-transfected with let-7a and Myc.6. Quantitative RT-PCR was used to test mature let-7a、pri-let-7a、up. pri-let-7a in U87 and U251 cells after transfected with si-HnRNPAlor HnRNPA1. 7. In U87 glioma cells xenograft model, the expression of c-Myc、HnRNPA1 and PKM2 were tested by immunohistochemistry.Results: 1. The lower expression of PKM2 significantly inhibited the growth of glioma cells and blocked G1 state of cell cycle. The concentration of glucose and OCR was increased; the concentration of lactate and ECAR was decreased. 2. Overexpression of let-7a inhibited the proliferation of glioma cells. In the let-7a mimics’ transfected cells, the concentration of glucose in culture medium and OCR was increased; the concentration of lactate culture medium and ECAR was decreased. Let-7a inhibites the expression of PKM2. 3. When si-hnRNPAl was transfected glioma cells, the expression of exon 9 and PKM1 was increased, while exon 10 and PKM2 was decreased, glucose metabolism experiment was similar to si-PKM2 or let-7a mimics transfected cells 4. C-Myc up-regulates transcription of hnRNPAl and bringing a high level of PKM2. Glucose metabolism experiment after treated with sh-c-MYC was similar to si-PKM2, let-7a mimics or si-hnRNPA1 transfected cells. HnRNPA1 plasmid largely abrogated the effect of sh-c-MYC on glucose metabolism in glioma cells. 5. In glioma cells, c-Myc is a functional target of let-7a that affects the glucose metabolism. And this effect does not appear after cotransfection of c-Myc. 6. HnRNPA1 block pri-let-7a-1 processing by Drosha and hinder the expression of let-7a. 7. Let-7a inhibites the expression of c-Myc, hnRNPA1 and PKM2 in U87 glioma cells xenograft model.Conclusions:1. Let-7a affects the growth and glucose metabolism of glioma cells via inhibiting the expression of PKM2.2. The let-7a, c-Myc and hnRNPAl from a feedback loop. This loop could regulate the expression of PKM2 to influences the growth and glucose metabolism of glioma cells.