Intestinal Lipid Metabolism Genes Regulated by miRNAs

Abstract

MicroRNAs (miRNAs) crucial roles in translation repression and post-transcriptional adjustments contribute to regulate intestinal lipid metabolism. Even though their actions in different metabolic tissues have been elucidated, their intestinal activity is yet unclear. We aimed to investigate intestinal miRNA-regulated lipid metabolism-related genes, by creating an intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mouse model, with a depletion of microRNAs in enterocytes. The levels of 83 cholesterol and lipoprotein metabolism-related genes were assessed in the intestinal mucosa of Int-Dicer1 KO and Wild Type C57BL/6 (WT) littermates mice at baseline and 2 h after an oral lipid challenge. Among the 18 genes selected for further validation, Hmgcs2, Acat1 and Olr1 were found to be strong candidates to be modulated by miRNAs in enterocytes and intestinal organoids. Moreover, we report that intestinal miRNAs contribute to the regulation of intestinal epithelial differentiation. Twenty-nine common miRNAs found in the intestines were analyzed for their potential to target any of the three candidate genes found and validated by miRNA-transfection assays in Caco-2 cells. MiR-31-5p, miR-99b-5p, miR-200a-5p, miR-200b-5p and miR-425-5p are major regulators of these lipid metabolism-related genes. Our data provide new evidence on the potential of intestinal miRNAs as therapeutic targets in lipid metabolism-associated pathologies.

Keywords: Acat1; Dicer1; Hmgcs2; Olr1; lipid metabolism; microRNA; organoids; small intestine.

FIGURE 1

(A) Effects of an oral high-fat dietary challenge (lipid challenge) on the gene expression of Dicer1 in small intestine of Wild Type C57BL/6 (WT) and intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mice. *p < 0.05 compared to WT+H₂O mice (n ≥ 23). (B) Dicer1 gene expression in intestinal organoids isolated from WT and Int-Dicer1 KO mice. *p < 0.05 compared to WT (n = 6). (C) DICER1 protein expression in small intestine scraped mucosa and intestinal organoids of WT and Int-Dicer1 KO mice. *p < 0.05 compared to WT group (n = 4). (D) Intestinal levels of miR-192 in WT and Int-Dicer1 KO mice. *p < 0.05 compared to WT+H₂O mice (n ≥ 23). (E) Effects of an oral lipid challenge on the expression of lipid and cholesterol metabolism-related genes, in small intestine from WT and Int-Dicer1 KO mice. Upregulated genes are represented in red; downregulated genes are shown in blue. (F) Effect of an oral lipid challenge on plasma triglyceride levels in WT and Int-Dicer1 KO mice. *p < 0.05 compared to WT+H₂O, ^#p < 0.05 compared to WT+lipid challenge mice (n ≥ 23). Data in panels (A–D,F) are means ± SEM. Comparison between groups by two-way ANOVA (A,F) or two-tailed unpaired t-tests (B–D).

FIGURE 2

(A) Effect of an oral high-fat dietary challenge (lipid challenge) on the expression of Abca1, Apoa4, ApoB, Apoc3, Acat1, Cnbp, Crp, Cyb5r3, Hmgcs2, Olr1 and Osbpl1, in small intestine of Wild Type C57BL/6 (WT) and intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mice. *p < 0.05 compared to WT+H₂O mice, ^#p < 0.05 compared to Int-Dicer1 KO+H₂O (n ≥ 23). (B) Acat1, Hmgcs2 and Olr1 gene expression in intestinal organoids isolated from WT and Int-Dicer1 KO mice exposed to postprandial micelles of olive oil and cholesterol (PPM). *p < 0.05 compared to control WT mice, ^#p < 0.05 compared to control Int-Dicer1 KO group (n = 9). (C) ACAT1, HMGCS2 and OLR1 protein expression in small intestine of WT and Int-Dicer1 KO mice. **p < 0.001, ***p < 0.0001, compared to WT (n = 9). (D) HMGCS2 and OLR1 protein expression in intestinal organoids isolated from WT and Int-Dicer1 KO mice. *p < 0.05, ***p < 0.0001 compared to WT (n = 3). Data in all cases are means ± SEM. Comparison between groups by two-way ANOVA (A,B) or two-tailed unpaired t-tests (C,D).

FIGURE 3

(A) Effect of an oral high-fat dietary challenge (lipid challenge) on the expression of Alpi, Chgb, Dpp4, Ki67, Gata4, Lyz1, Lgr5, Muc2, Slc2a2, Spp1, Tropp2 and Vill1, in small intestine of Wild Type C57BL/6 (WT) and intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mice. Data are means ± SEM. Comparison between groups by two-way ANOVA. *p < 0.05 compared to WT+H₂O mice (n ≥ 23). (B) Chga, Dpp4, Ki67, Gata4, Lyz1, Lgr5, Muc2, Slc2a2, Spp1, Tropp2 and Vill1 gene expression in intestinal organoids isolated from WT and Int-Dicer1 KO mice. Data are means ± SEM. Comparison between groups by two-tailed unpaired t-tests. *p < 0.05 compared to WT group (n = 9). (C) Light microscope visualization (10×) of mature intestinal organoids isolated from WT mice. (D) Light microscope visualization (10×) of mature intestinal organoids from Int-Dicer1 KO mice.

FIGURE 4

(A) PITA and TargetScan algorithms were run to predict 26 common miRNAs putatively regulating Hmgcs-2, Acat-1 and Olr-1. (B) miRNAs selected comparing the bioinformatic analysis with the list of 173 miRNAs expressed in intestinal epithelium.

FIGURE 5

Effects of several hsa-miRs on the expression levels of (A) Hmgcs2, (B) Acat1 and (C) Olr1, in Caco-2 cells transfected for 24 or 48 h. Data are means ± SEM. Comparison between groups by two-tailed unpaired t-tests. *p < 0.05 compared to control group. **p < 0.001 compared to control group (n = 4). (D) Effects of an oral high-fat dietary challenge (lipid challenge) on the expression of selected miRNAs, in small intestine of Wild Type C57BL/6 (WT) and intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mice. Data are means ± SEM. Comparison between groups by two-way ANOVA. *p < 0.05 compared to WT+H₂O mice (n ≥ 23).