engleski

„Omics“-approach to investigate cancer associated phenotypic changes in Hep G2 cells after targeted silencing of AHCY hydrolase

Most methylating enzymes use AdoMet as methyl group donor to methylate their substrate. AdoHyc is additional product of transmethylation reactions and is also proven to be one of the strongest competitive inhibitors of methyltransferases. Proper activity of AHCY hydrolase is therefore essential for fast removal of AdoHyc in order to avoid global alterations of cell methylation. Recently, it has been reported that level of AHCY hydrolase expression might have impact on changes in cell characteristics usually associated with so called cancer phenotype: cell cycle regulation, cell proliferation and migration. To investigate possible changes, targeted silencing of AHCY hydrolase in hepatocellular carcinoma cell line was performed using shRNA. Libraries for transcriptome sequencing were prepared automatically on NeoPrep Library System with TruSeq Stranded mRNA Library Prep Kit and sequenced on NextSeq desktop with integrated Basespace Sequence Hub platform (Illumina) for automatic data streaming, demultiplexing and filtering. Comparative proteomics was performed by Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and liquid chromatography – mass spectrometry (Orbitrap Velos Pro). All datasets were analyzed and compared using IPA software (QIAGEN) to investigate potential biomarkers and assess changes in cellular mechanisms, pathways and functions. Additionally, metabolic cell activity was measured by MTT viability test and Neutral red uptake assay to analyze differences in cell proliferation, while transwell chamber assay was used to study changes in cell migration. We found that both metabolic activity tests show decrease and indicate reduced cell proliferation. Also, slower cell migration is seen using transwell assay. IPA analysis confirmed these results and predicted that cell cycle is affected through several checkpoints leading to possible cell cycle arrest. Progression to cancer phenotype usually starts with alterations of cell cycle since its control is necessary for preventing unrestrained cellular growth. Our research indicates complex changes in regulation of cell cycle after AHCY hydrolase silencing with competing contribution of various pathways. Inhibitors of AHCY are currently being investigated as potential cancer treatments, thus understanding their effect on cell cycle is critical to predict possible treatment outcomes as well as for effective mixed drug therapies.

cancer, „omics“, cell cycle, methylation, AHCY hydrolase

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