Pyruvate Kinase M2(PKM2) Promotes Hepatocellular Cancer Development By Up-regulating Telomerase Reverse Transcriptase(hTERT) Expression

Objectives:Pyruvate kinase M2 (PKM2) plays an important role in malignant tumor metabolism, and provides tumor cells with enough energy and a lot of metabolic intermediate by mediating Warburg effect. hTERT is activated in most maliganant tumors and makes tumor cells immortalized. This study is aimed at Investigating the function of PKM2 in hepatocellular carcinoma (HCC), and the relationship between PKM2 and telomerase reverse transcriptase (hTERT).Methods:1. Cell proliferation test:After transfecting control or PKM2 siRNA into HCC HepG2 cells for 72 hours, we seeded 300 cells of each treatment group into 6-well-plates, and incubated them for 10 days. The cells were then stained with crystal violet, and cell clones were counted. After transfecting siRNA 24h/48h/72h, we determined cell viability using CCK-8 test.2. The relationship between PKM2 and hTERT.After transfecting control or PKM2 siRNA into HepG2 cells for 72 hours, we extracted total cellular RNA and proteins, and determined the mRNA levels of PKM2 or hTERT using qRT-PCR, protein level of PKM2 using western, and telomerase activity by ELISA. To investigate the effect of PKM2 knockdown on hTERT promoter activity, we constructed wild type hTERT promoter reporter plasmid. After transfecting control or PKM2 siRNA 24h, we transfected the renilla luciferase expression plasmid driven by a TK promoter and wild type hTERT promoter reporter plasmid, and then analyzed the firefly luciferase and renilla luciferase.3. The molecular mechanism underlying PKM2 regulation of hTERT.After transfecting control or PKM2 siRNA for 72h in HepG2 cells, we detected the mRNA and protein level of SP1. To investigate the binding of SP1 to the hTERT promoter after PKM2 knockdown, we performed Chromatin immunoprecipitation (ChIP) assay. We further mutated SP1 binding sites in hTERT promoter reporter plasmid. After transfecting siRNA for 24h, we transfected TK plasmid and hTERT promoter reporter plasmids with mutant Spl motifs, then determined promoter activities.4. After transfecting control or PKM1 siRNA into HepG2 cells for 72hrs, we collected cells. After transfecting vector or PKM1 overexpression plasmid 48h, we collected cells. We then extracted total cellular RNA and proteins, and determined the mRNA and protein level of PKM1. Meanwhile, we determined the mRNA level of hTERT、 Sp1、c-Myc and β-catenin, and the protein level of c-Myc and β-catenin.Results:1. PKM2 knockdown impairs cell clone formation ability. CCK-8 test confirmed that the number of viable cells in the PKM2 knockdown group was fewer than the control group, indicating that PKM2 knockdown weakens cell replicative capacity.2.PKM2 knockdown decreased the hTERT promoter activity、mRNA level of hTERT, and telomerase activity.3. PKM2 knockdown decreased the mRNA and protein level of the transcription factor SP1. SP1 binding site mutation partially reversed the effect of PKM2 knockdown on hTERT transcription. ChIP test indicated that PKM2 knockdown reduced the binding of SP1 protein on the hTERT promoter.4. hTERT mRNA level increased after PKM1 knockdown, coupled with the increased expression of c-Myc and P-catenin, while there was no significant change of Sp1.hTERT mRNA level decrease after PKM1 overexpression, the same as c-Myc and β-catenin.Conclusions:1. PKM2 promotes the proliferation of HepG2 cells.2. PKM2 positively regulates hTERT expression by enhancing the association of SP1 with the hTERT promoter.3. PKM1 may negatively regulates hTERT expression through the β-catenin-c-Myc axis.