Abstract
Cancer-specific isoenzyme of phosphofructokinase II (PFKFB4), as our previous research has shown, may be one of the most important enzymes contributing to the intensification of glycolysis in hypoxic malignant melanoma cells. Although the PFKFB4 gene seems to play a crucial role in the progression of melanoma, so far there are no complete data on the expression of PFKFB4 at the isoform level and the influence of hypoxia on alternative splicing. Using RT-qPCR and semi-quantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Additionally, using VMD software, we analyzed the structure of isoforms at the protein level, concluding about the catalytic activity of individual isoforms. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our results also indicate that the expression profile at the level of the PFKFB4 gene does not reflect the expression at the level of individual isoforms. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. Moreover, the analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells.
Highlights
Jagiellonian University Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Jagiellonian University Medical College, Center for Medical Genomics-OMICRON, 31-034 Krakow, Poland; Academic Editor: Beate Heissig
As hypoxia-inducible factor (HIF)-1 can be considered as the primary mediator of the adaptive response of cells to hypoxia, we have examined the impact of low oxygen concentration on the stabilization of HIF-1 alpha subunit protein
Our analysis shows that hypoxia does not significantly regulate PFKFB4 expression at the protein level, while at the mRNA level the response is much weaker than for CAIX
Summary
Jagiellonian University Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Jagiellonian University Medical College, Center for Medical Genomics-OMICRON, 31-034 Krakow, Poland; Academic Editor: Beate Heissig. Using RT-qPCR and semiquantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. The analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells. Epigenetic, and functional changes in cancer cells are crucial for the process of published maps and institutional affiliations
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