Trimethylamine n-Oxide (TMAO) Modulates the Expression of Cardiovascular Disease-Related microRNAs and Their Targets

Abstract

Diet is a well-known risk factor of cardiovascular diseases (CVDs). Some microRNAs (miRNAs) have been described to regulate molecular pathways related to CVDs. Diet can modulate miRNAs and their target genes. Choline, betaine, and l-carnitine, nutrients found in animal products, are metabolized into trimethylamine n-oxide (TMAO), which has been associated with CVD risk. The aim of this study was to investigate TMAO regulation of CVD-related miRNAs and their target genes in cellular models of liver and macrophages. We treated HEPG-2, THP-1, mouse liver organoids, and primary human macrophages with 6 µM TMAO at different timepoints (4, 8, and 24 h for HEPG-2 and mouse liver organoids, 12 and 24 h for THP-1, and 12 h for primary human macrophages) and analyzed the expression of a selected panel of CVD-related miRNAs and their target genes and proteins by real-time PCR and Western blot, respectively. HEPG-2 cells were transfected with anti-miR-30c and syn-miR-30c. TMAO increased the expression of miR-21-5p and miR-30c-5p. PER2, a target gene of both, decreased its expression with TMAO in HEPG-2 and mice liver organoids but increased its mRNA expression with syn-miR-30c. We concluded that TMAO modulates the expression of miRNAs related to CVDs, and that such modulation affects their target genes.

Keywords: TMAO; atherosclerosis; cardiovascular disease; epigenetics; miRNAs; nutrition; target genes.

Figure 1

TMAO effect on the expression of miR-21-5p and miR-30c-5p in HEPG-2 and THP-1 cells. HEPG-2 (a) and THP-1 (b) cells were treated with 6 µM TMAO for 4, 8, and 24 h (HEPG-2) and 12 h and 24 h (THP-1). miR-21-5p (black bars) and miR-30c-5p (light grey bars) levels were measured by RT-qPCR. miRNA relative expression levels were calculated using the 2^−ΔΔCT method comparing treated cells with each corresponding untreated control sample at each timepoint. The mean ± SEM of three independent experiments is shown. Dashed lines represent the no-change threshold. * p < 0.05 for the comparison of each timepoint with the corresponding untreated timepoint using a Student’s t-test.

Figure 2

TMAO effect on the expression of miR-21-5p and miR-30c-5p in mouse liver organoids and human primary macrophages. Mouse liver organoids (a) and human primary macrophages (b) were treated with 6 µM TMAO for 4, 8, and 24 h (liver organoids) and for 12 h (human primary macrophages). miR-21-5p (black bars) and miR-30c-5p (light-gray bars) levels were measured by RT-qPCR. miRNA relative expression levels were calculated using the 2^−ΔΔCT method, comparing treated cells with each corresponding untreated control sample at each timepoint. The mean ± SEM of three independent experiments is shown. Dashed lines represent the no-change threshold. * p < 0.05 for the comparison of each timepoint with the corresponding untreated timepoint using a Student’s t-test.

Figure 3

Effect of TMAO on PER2 gene and protein expression levels. Cells were treated with 6 µM TMAO for 8 h (black bars) and 24 h (gray bars), and gene (a) and protein (b) expression levels of PER2 were measured in HEPG-2 cells. Gene expression was measured by RT-qPCR and calculated using the 2^−ΔΔCT relative expression method, comparing treated cells with each corresponding untreated control sample at each timepoint. Protein levels were measured by Western blot. Band density was measured and normalized with β-actin. The mean ± SEM of three independent experiments is shown. Dashed lines represent the no-change threshold. * p < 0.05 for the comparison of each timepoint with the corresponding untreated timepoint using a Student’s t-test.

Figure 4

Effect of miR-30c inhibition or overexpression on TMAO-mediated regulation of miR-30c-5p in HEPG-2 cells. Cells were transfected with 50 nM anti-miRNA-30c or 5 nM syn-miRNA-30c and incubated with 6 µM TMAO for 4 (black bars), 8 (dark-gray bars), and 24 h (light-gray bars). The expression of miR-30c-5p, was measured by RT-qPCR. mRNA levels were calculated using the 2^−ΔΔCT relative method, comparing with mock-transfected cells (blank). The mean ± SEM of three independent experiments is shown. * p < 0.05 comparing each untreated condition with its corresponding TMAO-treated condition using a Student’s t-test; + p < 0.05, ++ p < 0.001 comparing anti-miR-30c- and syn-miR-30c-transfected samples with their corresponding non-transfected samples using a Student’s t-test.

Figure 5

Effect of miR-30c inhibition or overexpression on TMAO-mediated regulation of PER2 in HEPG-2 cells. Cells were transfected with 50 nM anti-miRNA-30c or 5 nM syn-miRNA-30c and incubated with 6 µM TMAO for 4 (black barks), 8 (dark-gray bars), and 24 h (light-gray bars). The expression of PER2 was measured by RT-qPCR. mRNA levels were calculated using the 2^−ΔΔCT method. The mean ± SEM of three independent experiments is shown. * p < 0.05, comparing each untreated condition with its corresponding TMAO-treated condition using a Student’s t-test; + p < 0.05, ++ p < 0.001 comparing anti-miR-30c- and syn-miR-30c-transfected samples with their corresponding non-transfected sample using a Student’s t-test.