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. 2017 Nov 2;2(21):e96094.
doi: 10.1172/jci.insight.96094.

Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH

Melanie Tran  1 , Sang-Min Lee  1 , Dong-Ju Shin  1 , Li Wang  1   2   3   4
Affiliations

Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH

Melanie Tran et al. JCI Insight. .

Abstract

Accumulation of lipid droplets and inflammatory cell infiltration is the hallmark of nonalcoholic steatohepatitis (NASH). The roles of noncoding RNAs in NASH are less known. We aim to elucidate the function of miR-141/200c in diet-induced NASH. WT and miR-141/200c-/- mice were fed a methionine and choline deficient (MCD) diet for 2 weeks to assess markers of steatosis, liver injury, and inflammation. Hepatic miR-141 and miR-200c RNA levels were highly induced in human patients with NASH fatty liver and in WT MCD mice. miR-141/200c-/- MCD mice had reduced liver weights and triglyceride (TG) levels, which was associated with increased microsomal TG transfer protein (MTTP) and PPARα but reduced SREBP1c and FAS expression. Inflammation was attenuated and F4/80 macrophage activation was suppressed in miR-141/200c-/- mice, as evidenced by decreased serum aminotransferases and IL-6 and reduced hepatic proinflammatory, neutrophil, and profibrotic genes. Treatment with LPS in BM-derived macrophages isolated from miR-200c/141-/- mice polarized macrophages toward the M2 antiinflammatory state by increasing Arg1 and IL-10 levels while decreasing the M1 marker iNOS. In addition, elevated phosphorylated AMPK (p-AMPK), p-AKT, and p-GSK3β and diminished TLR4 and p-mTOR/p-4EBP1 proteins were observed. Lipidomics and metabolomics revealed alterations of TG and phosphatidylcholine (PC) lipid species by miR-141/200c deficiency. In summary, miR-141/200c deficiency diminished NASH-associated hepatic steatosis and inflammation by reprogramming lipid and inflammation signaling pathways.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Levels of miR-141/200c are increased in steatotic livers of patients and mice with NASH.
(A) qPCR analysis of miR-141 and miR-200c expression in human liver specimens (n = 10–20 samples per group). (B) qPCR analysis of miR-141 and miR-200c expression in livers of WT mice fed the control diet (CD) and methionine and choline–deficient (MCD) diet (n = 5–7 mice per group). (C) Targeting strategy for generating miR-141/200c–double KO mice. (D) qPCR analysis of miR-141 and miR-200c expression in livers of miR-141/200c–/– mice and their WT littermates (n = 5 mice per group). Data are represented as mean ± SEM. Differences between 2 groups were compared using a Student’s unpaired t test. For multiple groups, differences were compared using a one-way ANOVA followed by Newman-Keuls multiple comparisons test. **P < 0.01 indicate statistical significance.
Figure 2
Figure 2. MCD diet–induced steatosis is lessened by miR-141/200c deficiency.
(A) Body weight, absolute and relative liver weights in WT and miR-141/200c–/– mice fed the control diet (CD) or methionine and choline–deficient (MCD) diet (n = 4–8 mice per group). (B) Serum and liver triglyceride levels were measured by calorimetric analysis (n = 4–8 mice per group). (C) Representative images of H&E staining of liver sections from WT and miR-141/200c–/– mice fed the CD and MCD diet. (D and E) Western blot showing liver protein levels of pSREBP1, nSREPB1, FAS, MTTP, and α-TUBULIN in WT MCD and miR-141/200c–/– MCD mice. Samples were pooled from 5 individual mice for each group unless otherwise indicated. Densitometry of each blot relative to the loading control was quantified using ImageJ software. Data are represented as mean ± SEM. Differences between 2 groups were compared using a Student’s unpaired t test. For multiple groups, differences were compared using a one-way ANOVA followed by Newman-Keuls multiple comparisons test. *P < 0.05 and **P < 0.01 indicate statistical significance.
Figure 3
Figure 3. MCD diet–induced hepatic inflammation is diminished by miR-141/200c deficiency.
(A) Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT),and (B) IL-6 were measured in overnight fasted serum of WT and miR-141/200c–/– mice fed the control diet (CD) or methionine and choline–deficient (MCD) diet (n = 5–8 mice per group). (C–F) qPCR analysis of hepatic genes involved in inflammation (Saa1, Orm1, IL-1β, TNFα, IL-4, F4/80, LyG6) and fibrosis (col1a1, TGFβ) (n = 5 mice per group). (G) Representative images of Masson’s trichrome staining and F4/80 immunohistochemistry to examine fibrosis and macrophage activation in WT MCD and miR-141/200c–/– MCD. Images were quantified by digital image analysis using ImageJ software in 6 randomly chosen fields from 3 individual mice per group. (H) qPCR analysis of hepatic genes associated with M1 (iNOS) or M2 (Arg1) macrophage phenotype in WT MCD and miR-141/200c–/– MCD mice (n = 5 mice per group). Data are represented as mean ± SEM. Differences between 2 groups were compared using a Student’s unpaired t test. For multiple groups, differences were compared using a one-way ANOVA followed by Newman-Keuls multiple comparisons test. *P < 0.05 and **P < 0.01 indicate statistical significance.
Figure 4
Figure 4. miR-141/200c deficiency polarizes macrophages toward the M2 antiinflammatory phenotype.
BM-derived macrophages (BMDMs) were generated from WT and miR-141/200c–/– mice and incubated with or without LPS (10 ng/ml) for 24 hours and then processed for RNA isolation (n = 2 independent experiments). qPCR analysis of mRNA expression of (A) M1 markers (iNOS, IL-6, and IL-1β), (B) M1 regulators (HIF1α and NF-κβ), (C) M2 markers (Arg1 and IL-10), and (D) M2 regulators (KLF4, C/EBPβ, and STAT3). Data are represented as mean ± SEM. Differences between multiple groups were compared using a one-way ANOVA followed by Newman-Keuls multiple comparisons test. *P < 0.05 and **P < 0.01 indicate statistical significance.
Figure 5
Figure 5. miR-141/200c deficiency affects proteins in multiple signaling pathways.
Immunoblotting analysis of proteins in (A) PPAR and AMPK; (B) ERK, SAPK/JNK, and p38; (C) AKT/GSK3β; (D) TLR4; and (E) mTOR signaling pathways in WT and miR-141/200c–/– livers fed the MCD diet. Samples were pooled from 5 individual mice for each group. Densitometry of each blot relative to the loading control was quantified using ImageJ software.
Figure 6
Figure 6. Lipidomics reveals reduced TG and increased PC lipid species in miR-141/200c–/– mice.
Livers of WT and miR-141/200c–/– mice from the methionine and choline–deficient (MCD) group were prepared and subjected to LC-MS for lipidomics analysis of various lipid classes (n = 5 mice per group). (A) Triglyceride (TG), (B) phosphatidylcholine (PC), (C) phospholipids including phosphatidylinositol (PI) and phosphatidylserine (PS), (D) sphingomyelin (SM), and (E) ceramide lipid species are expressed as the fold change relative to WT mice. Data are represented as mean ± SEM. *P < 0.05 and **P < 0.01 vs. WT by Student’s unpaired t test.
Figure 7
Figure 7. Metabolomics unravels distinct metabolic profiling altered by miR-141/200c deficiency.
Livers of WT and miR-141/200c–/– mice from the methionine and choline–deficient (MCD) group were prepared and subjected to LC-MS for metabolomics analysis (n = 5 mice per group). Levels of (A) amino acids, (B) saturated fatty acids, and (C) cholic acid are expressed as the fold change relative to WT mice. Data are represented as mean ± SEM. *P < 0.05 vs. WT by a Student’s unpaired t test. (D) Schematic diagram of miR-141/200c–modulated signaling pathways to control lipid metabolism and macrophage phenotype in MCD diet–induced NASH. miR-141/200c inhibits AMPK phosphorylation to reduce downstream PPARα activation and subsequently decrease fatty acid β-oxidation. At the same time, miR-141/200c targets mTOR phosphorylation to favor SREPB1 translocation to the nucleus, contributing to hepatic lipid accumulation. In the presence of LPS, miR-141/200c deficiency appears to favor antiinflammatory M2 polarization and activation as assessed by the increased expression of Arg1 and IL-10.
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