Integrated transcriptomic and proteomic analyses reveal ɑ-lipoic acid-regulated cell proliferation via Grb2-mediated signalling in hepatic cancer cells

Abstract

Hepatocellular carcinoma is the most frequent primary liver cancer worldwide. The use of antioxidants as cancer prevention and treatment agents has become a focus of research in recent years due to their limited adverse effects. Alpha lipoic acid (ɑ-LA) is synthesized in the liver and is considered a naturally occurring antioxidant. In this study, a total of 4446 differentially expressed genes (2097 down-regulated and 2349 up-regulated) were identified via RNA-Seq in HepG2 cells after exposure to α-LA for 24 hrs. Moreover, GO and KEGG pathway analyses showed that cancer-relevant cell membrane proteins were significantly affected. An interaction network analysis predicted that Grb2 might mediate the key target pathways activated by exposure to ɑ-LA. Verification of the RNA-Seq and iTRAQ results confirmed that Grb2 mediated the ɑ-LA-induced inhibition of cell proliferation in vitro. Furthermore, the analysis of human hepatocellular carcinoma specimens obtained from the GEO database showed that the expression of EGFR and Met correlated with that of Grb2. These findings provide a novel mechanism through which ɑ-LA regulates cell proliferation via the down-regulation of growth factor-stimulated Grb2 signalling.

Keywords: Grb2; RNA-Seq; hepatocellular carcinomas; proteome; ɑ-lipoic acid.

Figure 1

Transcriptome profiling in response to 2 mM α‐LA treatment. (A) Heatmap revealing the differentially expressed genes in three replicates of the 24‐hr control‐ and α‐LA‐treated HepG2 cells. Each row represents the relative expression levels of a single gene across all samples. The red blocks represent high expression relative to the control cells, and blue blocks represent low relative expression. (B and C) GO and KEGG enrichment analysis of the DEGs in HepG2 cells after α‐LA treatment. This analysis was performed using the online tool DAVID (https://david.ncifcrf.gov/).

Figure 2

Functional classification of α‐LA‐responsive proteins in HepG2 cells. The proteins were classified into categories for (A) molecular function and (B) cellular component. (C) KEGG pathway analysis of DEP enrichment in HepG2 cells after α‐LA treatment.

Figure 3

Protein–protein interaction network of DEPs. (A) Venn diagram showing the overlapping DEGs (blue) and DEPs (yellow). (B) Heatmaps showing the expression variation at the mRNA level of the 72 DEGs/DEPs. (C) Protein–protein interactions among these 72 DEGs/DEPs according to the STRING database (http://string-db.org/). Line segments indicate protein–protein interactions, red indicates up‐regulated DEGs/DEPs, and green indicates down‐regulated DEGs/DEPs. (D) Grb2‐centred protein–protein interaction network. The network was constructed with the BioGRID database (https://thebiogrid.org/) and used to predict the protein network related to Grb2.

Figure 4

Grb2 mediates the ɑ‐LA‐induced reduction in cell proliferation. (A) HepG2 cell proliferation was measured through a CCK‐8 assay at the indicated times. (B and C) Cell cycle regulatory protein expression in HepG2 cells after treatment with 2.0 mM ɑ‐LA for 24 hrs was assessed via Western blotting. (D) Grb2 levels were measured via real‐time PCR and Western blotting in HepG2 cells after treatment with 1.0 mM ɑ‐LA for 12 and 24 hrs (upper panel). HepG2 cells were transfected with siRNA against Grb2, and 24 hrs after transfection, the cells were seeded into 96‐well plates for CCK‐8 assays at the indicated times (middle panel). After transfection with Grb2 overexpression plasmids, cell proliferation was measured at the indicated times through a CCK‐8 assay (lower panel). (E) A Western blotting assay was performed to assess the levels of phosphorylated EGFR, Met, ERK and Ras in HepG2 cells after treatment with 2.0 mM ɑ‐LA for 6 and 24 hrs. (F) HepG2 cells were transfected with 50 nM scramble siRNA or Grb2 siRNA (siGrb2) for 48 hrs and then treated with ɑ‐LA for 24 hrs, and the levels of phosphorylated EGFR and Met were analysed via Western blotting.

Figure 5

Correlations of EGFR, Met and Grb2 in human HCC specimens. (A) Grb2 expression in normal tissue downloaded from the GeneCards website. (B) Grb2 expression in various cancers queried from the cBioPortal database. (C) The expression of Grb2 in HCC and adjacent tissue (NCBI GEO accession number 20140) was analysed. (D) Kaplan–Meier curves showing survival times according to the Grb2 signature in HCC patients. The patients with expression above the median are shown in red, and the patients with expression below the median are shown in blue. (E) A Pearson's correlation analysis of Met/Grb2 and EGFR/Grb2 expression from microarrays of human hepatic malignant lesions tissue (NCBI GEO accession number 20140), which provided data from 287 specimens, including a training cohort (80 tumour and 82 non‐tumour liver samples) and a validation cohort (225 non‐tumour liver tissue samples surgically resected from patients with HCC), was performed.

Figure 6

Schematic diagram illustrating how ɑ‐LA, via regulation of Grb2 expression, modulates different signalling pathways that participate in proliferation‐related stress responses. Growth factors (EGF/HGF) in the extracellular matrix bind to RTKs on the plasma membrane and subsequently phosphorylate the docking site and recruit effector molecules [Grb2, Grb2‐associated‐binding protein 1 (Gab 1), SRC homology 2 domain‐containing phosphatase 2 (SHP2), Son of Sevenless (Sos) and sarcoma non‐receptor tyrosine kinase (SRC)]. ɑ‐LA, by down‐regulating Grb2 expression, impairs the phosphorylation of the docking site of effector molecules and then subsequently attenuates the downstream ERK/MAPK pathway and the PI3K‐AKT pathway, leading to decreased ERK1/2 and/or mTOR translocation across the nuclear membrane and/or inhibition of the activation of transcription factors (TFs) and gene expression. The signals affect gene expression and repress cell proliferation and survival, resulting in the arrest of cancer growth and progression.