Resveratrol and EGCG bind directly and distinctively to miR-33a and miR-122 and modulate divergently their levels in hepatic cells

Abstract

Modulation of miR-33 and miR-122 has been proposed to be a promising strategy to treat dyslipidemia and insulin resistance associated with obesity and metabolic syndrome. Interestingly, specific polyphenols reduce the levels of these mi(cro)RNAs. The aim of this study was to elucidate the effect of polyphenols of different chemical structure on miR-33a and miR-122 expression and to determine whether direct binding of the polyphenol to the mature microRNAs (miRNAs) is a plausible mechanism of modulation. The effect of two grape proanthocyanidin extracts, their fractions and pure polyphenol compounds on miRNA expression was evaluated using hepatic cell lines. Results demonstrated that the effect on miRNA expression depended on the polyphenol chemical structure. Moreover, miR-33a was repressed independently of its host-gene SREBP2. Therefore, the ability of resveratrol and epigallocatechin gallate to bind miR-33a and miR-122 was measured using (1)H NMR spectroscopy. Both compounds bound miR-33a and miR-122 and differently. Interestingly, the nature of the binding of these compounds to the miRNAs was consistent with their effects on cell miRNA levels. Therefore, the specific and direct binding of polyphenols to miRNAs emerges as a new posttranscriptional mechanism by which polyphenols could modulate metabolism.

Figure 1.

Chemical structure of representative polyphenols.

Figure 2.

Effect of polyphenolic extracts, pure compounds and microbial metabolites on the levels of miR-33a and miR-122 in rat hepatoma Fao cells. Fao cells were treated with 25 mg/l of polyphenolic extracts and fractions (A, B), 50 μM of pure compounds (C, D) and 50 μM of microbial metabolites (E, F) for 1 h. miRNA levels were determined by qRT-PCR and normalized to U6 snRNA levels. All values represent the mean of three independent experiments. White bars represent the control group (CNT), and black bars represent the treated group. An asterisk denotes a significant difference between control cells and treated cells (P < 0.05; Student’s t-test). Abbreviations: CNT, control cells; GSPE, grape seed proanthocyanidin extract; GPE, grape pomace extract; MF, monomeric fraction; DF, dimeric fraction; OF, oligomeric fraction; Q, quercetin; C, catechin; EC, epicatechin; RSV, resveratrol; EGCG, epigallocatechin gallate; B2, dimer B2; HBA, 3′-hydroxybenzoic acid; m-HPAA, m-hydroxyphenylacetic acid; VA, vanillic acid; and GA, gallic acid.

Figure 3.

Effect of polyphenolic extracts, pure compounds and microbial metabolites on the levels of miR-33a and miR-122 in human hepatoma HepG2 cells. HepG2 cells were treated with 25 mg/l of polyphenolic extracts and fractions (A, B), 50 μM of pure compounds (C, D) and 50 μM of microbial metabolites (E, F) for 1 h. Experimental details, symbols and abbreviations are as in Figure 2.

Figure 4.

Effect of RSV, EGCG, Q and GA on the mRNA and protein levels of FAS in HepG2 cells. HepG2 cells were treated with 50 μM of RSV (A), EGCG (B), Q (C) and GA (D) for 1 h to analyze FAS mRNA levels and for 5 h to analyze FAS protein levels. mRNAs levels were determined by qRT-PCR and normalized to PPIA mRNA levels. Proteins were extracted with RIPA buffer and analyzed by Western blot. Protein levels were normalized to an endogenous protein, Hsp90. Relative intensity units were obtained by dividing the intensity of the band of the target protein by that of the endogenous protein. All of the values represent the means of two independent experiments. An asterisk denotes a significant difference between control cells and treated cells (P < 0.05; Student’s t-test). Abbreviations: EGCG, epigallocatechin gallate; GA, gallic acid; Q, quercetin; and RSV, resveratrol.

Figure 5.

Effect of RSV, EGCG, Q and GA on the mRNA and protein levels of ABCA1 in HepG2 cells. HepG2 cells were treated with 50 μM of RSV (A), EGCG (B), Q (C) and GA (D) for 1 h to analyze ABCA1 mRNA levels and for 5 h to analyze ABCA1 protein levels. Experimental details, symbols and abbreviations are as in Figure 4.

Figure 6.

SREBP2 mRNA levels after 1 h RSV, EGCG, Q and GA treatment in HepG2 cells. HepG2 cells were treated with 50 μM of RSV, EGCG, Q and GA for 1 h to analyze mRNA levels of SREBP2. mRNAs levels were determined by RTqPCR and normalized to PPIA mRNA levels. All the values are the means of three independent experiments. Asterisk denotes significant difference between control cells and treated cells (P < 0.05; Student’s t-test). Abbreviations: RSV, resveratrol; EGCG, epigallocatechin gallate; Q, quercetin; GA, gallic acid.

Figure 7.

¹H NMR spectra of RSV and EGCG in the presence of miR-33a and miR-122. (A) The chemical structure of RSV. (B) ¹H NMR spectra of RSV alone or in solution with mature miR-33a. (C) ¹H NMR spectra of RSV alone or in solution with mature miR-122. (D) The chemical structure of EGCG. (E) ¹H NMR spectra of EGCG alone or in solution with mature miR-33a. (F) ¹H NMR spectra of EGCG alone or in solution with mature miR-122. The miRNA concentration was 50 µM, and the concentrations of the polyphenols were 15, 30 and 50 µM to obtain the polyphenol:miRNA ratios of 0.3:1, 0.6:1 and 1:1, respectively. Abbreviations: EGCG, epigallocatechin gallate; and RSV, resveratrol.