Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells

Abstract

Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC-MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson's disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency.

Figure 1. Effect of riboflavin on cell viability, apoptosis and glutathione reductase activity.

The riboflavin adequate group (12.76 nmol/L) was defined as the control group. (A) cell viability. (B) cell apoptosis. (C) glutathione reductase activity.

Figure 2. Hierarchical cluster of proteins differentially expressed between riboflavin deficient (test 1–3) and riboflavin adequate (control 1–3) samples with an FDR < 1% identified by MaxQuant.

Red, high expression; green, low expression. Two main clusters of proteins can be observed, one up-regulated (right) and other down-regulated (left) in riboflavin deficient group. Protein position in the cluster can be found in Table 1.

Figure 3. KEGG Pathway analysis.

KEGG pathway enrichment analyses were applied based on the Fisher’ exact test, considering the whole quantified protein/phosphorproteins annotation as background dataset. Benjamini- Hochberg correction for multiple testing was further applied to adjust derived p-values. Only pathways with p-values under a threshold of 0.05 were considered as significant.

Figure 4. Protein–protein interaction analysis performed by InAct.

Differentially expressed proteins are highlighted in yellow.

Figure 5. Immunoblotting validation of differentially expressed proteins.

(A) NDUFS1, SDHA, NDUFV2, ERO1A and SQSTM1 were selected to be validated by immunoblotting test (β-actin as the control). (B) quantification of immunoreactive band density measured in Panel A. Data are presented as the percent change relative to control (12.76 nmol/L) samples. The blots/gels were cropped and full-length blots/gels are presented in Supplementary Fig. S1. Data are presented as mean fold values ± SD of the changed expression at the protein level. *p < 0.01.