RNA-seq data analysis of stimulated hepatocellular carcinoma cells treated with epigallocatechin gallate and fisetin reveals target genes and action mechanisms

Panagiotis C Agioutantis; Vasilios Kotsikoris; Fragiskos N Kolisis; Heleni Loutrari

doi:10.1016/j.csbj.2020.03.006

RNA-seq data analysis of stimulated hepatocellular carcinoma cells treated with epigallocatechin gallate and fisetin reveals target genes and action mechanisms

Comput Struct Biotechnol J. 2020 Mar 18:18:686-695. doi: 10.1016/j.csbj.2020.03.006. eCollection 2020.

Authors

Panagiotis C Agioutantis^{1

2}, Vasilios Kotsikoris¹, Fragiskos N Kolisis², Heleni Loutrari¹

Affiliations

¹ G.P. Livanos and M. Simou Laboratories, 1st Department of Critical Care Medicine & Pulmonary Services, Evangelismos Hospital, Medical School, National Kapodistrian University of Athens, 3 Ploutarchou Str., Athens 10675, Greece.
² Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.

Abstract

Hepatocellular carcinoma (HCC) is an essentially incurable inflammation-related cancer. We have previously shown by network analysis of proteomic data that the flavonoids epigallocatechin gallate (EGCG) and fisetin (FIS) efficiently downregulated pro-tumor cytokines released by HCC through inhibition of Akt/mTOR/RPS6 phospho-signaling. However, their mode of action at the global transcriptome level remains unclear. Herein, we endeavor to compare gene expression alterations mediated by these compounds through a comprehensive transcriptome analysis based on RNA-seq in HEP3B, a responsive HCC cell line, upon perturbation with a mixture of prototypical stimuli mimicking conditions of tumor microenvironment or under constitutive state. Analysis of RNA-seq data revealed extended changes on HEP3B transcriptome imposed by test nutraceuticals. Under stimulated conditions, EGCG and FIS significantly modified, compared to the corresponding control, the expression of 922 and 973 genes, respectively, the large majority of which (695 genes), was affected by both compounds. Hierarchical clustering based on the expression data of shared genes demonstrated an almost identical profile in nutraceutical-treated stimulated cells which was virtually opposite in cells exposed to stimuli alone. Downstream enrichment analyses of the co-modified genes uncovered significant associations with cancer-related transcription factors as well as terms of Gene Ontology/Reactome Pathways and highlighted ECM dynamics as a nodal modulation point by nutraceuticals along with angiogenesis, inflammation, cell motility and growth. RNA-seq data for selected genes were independently confirmed by RT-qPCR. Overall, the present systems approach provides novel evidence stepping up the mechanistic understanding of test nutraceuticals, thus rationalizing their clinical exploitation in new preventive/therapeutic modalities against HCC.

Keywords: ADAM, a disintegrin and metalloproteinase with thrombospondin motifs; ADAMTS9, ADAM metallopeptidase with thrombospondin type 1 motif 9; CLIC3, Chloride Intracellular Channel 3; CTGF, Connective Tissue Growth Factor; DEGs, differentially expressed genes; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; EGCG, epigallocatechin gallate; EMT, epithelial to mesenchymal transition; Epigallocatechin gallate; FIS, fisetin; Fisetin; GO, Gene Ontology; Gene Ontology; HCC, hepatocellular carcinoma; HSPA2, Heat Shock Protein Family A (Hsp70) Member 2; HSPB1, Heat Shock Protein Family B (Small) Member 1; Hepatocellular carcinoma; MEM, minimum essential medium; MMP11, Matrix Metallopeptidase 11; MMP9, Matrix Metallopeptidase 9; MMPs, matrix metalloproteinases; PDGFRB, Platelet Derived Growth Factor Receptor Beta; RNA-sequencing; RT-qPCR, reverse transcription-quantitative real time PCR; Reactome Pathways; SD, standard deviation; SEM, standard error of mean; SERPINE1, Serpin Family E Member 1; STIM, stimulated; TF, transcription factor; Transcription factors.