Translational Regulation of Glycolytic Enzymes in Cancer

Abstract

Aerobic glycolysis is the hallmark of many cancer cells, which results in high rate of ATP production and more importantly the biosynthetic intermediates that are required by the fast growing tumor cells. The molecular mechanism responsible for the increased glycolytic influx of tumor cells is still not fully understood. In the present study, we tried to address the newlineabove question by exploring the role of the glycolytic enzymes, triose phosphate isomerase (TPI) and pyruvate kinase (PKM) in the cancer cells. The Western blot analysis of the human colorectal samples depicted higher post-transcriptional expression of TPI and PKM in the tumor relative to the normal tissue. In-silico analysis indicated that the TPI mRNA newlinepossess putative binding site for the miR-22, miR-23, miR-28 and miR-140. Similarly, PKM mRNA was depicted to possess the binding sites for the miR-149, miR-338, miR-612 and miR-762. Using various biochemical assays, only miR-22 and miR-28 were confirmed to bind the 3`UTR of the TPI mRNA and regulate its expression. The specificity of the miR-22/28 regulation of the TPI mRNA was confirmed using dual luciferase assay (DLR) and mutagenic experiments. Moreover, the hypoxia conditions resulted in the increased newlineexpression of the TPI protein, with concomitant decrease of the miR-22/28. The newlinephysiological significance of the TPI and miR-22/28 interaction on the glycolytic influx was confirmed by the L-lactate production in the HCT-116++ cells. Additionally, the 5`UTR of TPI and PKM mRNAs were cloned in the bi-cistronic vectors to establish whether any cap-independent translation plays role in the post-transcriptional regulation of these glycolytic enzymes. Our results demonstrated that the TPI 5`UTR possess a putative internal ribosome newlineentry site (IRES) element that may be responsible for the cap-independent translation of TPI newlinemRNA. However, no nucleotide difference was observed in the 5`UTR of the TPI mRNAs newlineviz-a-viz colorectal cancer tissue and that of the adjacent normal samples. Overall, our data ......