Elevated miR-34a induced by lipotoxicity and inflammation mediates pathophysiological communication between hepatocytes and hepatic stellate cells in liver fibrosis

Abstract

Increased mortality in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) imposes an urgent need to elucidate the pathogenesis of MASLD so that novel therapeutic strategies may be identified. Here, we delineate the mechanism of microRNA-34a-5p (miR-34a) in the progressive liver injury of MASLD and liver fibrosis. Specifically, liver tissue from patients with obesity-associated hepatic steatosis, metabolic dysfunction-associated steatohepatitis (MASH), and fibrosis, as well as liver tissues from a human MASLD-like mouse model, were utilized for this study. We found that lipotoxicity resulting from obesity or saturated free fatty acid treatment induced miR-34a expression in human liver tissue or mouse hepatocytes, which was accompanied by dysregulation of lipoprotein metabolism, activation of inflammation, and ballooning degeneration of hepatocytes. Moreover, increased cellular miR-34a induced by treatment with saturated fat, palmitic acid, or transfection of miR-34a mimic was released from injured hepatocytes into the conditional cell culture media, which mediated pathological communications between hepatocytes and hepatic stellate cells, activated pro-fibrogenic signaling in hepatic stellate cells, and induced extracellular matrix remodeling. These phenotypes were recapitulated in a human MASLD-like mouse model in which MASLD and liver fibrosis were induced via streptozotocin treatment and high-fat feeding. Elevated expression of miR-34a was found in mouse liver tissues, which conveyed the progressive liver injury from steatosis to MASH and liver fibrosis. Our findings demonstrate that elevated miR-34a induced by lipotoxicity and metabolic inflammation are key driving factors in the progressive liver injury from simple steatosis to MASH and liver fibrosis.

Keywords: Cell–cell communication; Hepatic stellate cells; Liver fibrosis; MASLD; microRNA-34a.

Figure 1

Elevated miR-34a induced by lipotoxicity-activated inflammatory signaling in hepatocytes. (A) AML12 cells were treated with Mock (0.5% BSA)/Veh (H₂O), PA (0.25 mM)/Veh (H₂O), or TNFα (20 ng/mL)/Mock (0.5% BSA) for 18 h mRNA expression of miR-34a was detected with qRT-PCR. (B) Image of miR-34a fluorescent in situ hybridization (FISH) signals in AML12 cells treated with Mock (0.5% BSA)/Veh (H₂O), PA (0.25 mM)/Veh (H₂O), or TNFα (20 ng/mL)/Mock (0.5% BSA) for 18 h. Scale bar, 10 μm. (C) mRNA expression of lipoproteins ApoB, ApoE, and Mttp by qRT-PCR in AML12 cells transfected with Scram (control) or miR-34a mimics for 48 h. (D) TG in AML12 cells transfected with Scram or miR-34a mimics for 48 h. (E) Immunoblotting analysis of GRP78, eIF2α-p, JNK-p, and loading control β-actin protein in the AML12 cells transfected with Scram or miR-34a mimics for 48 h. (F) mRNA expression of Tnfα and Il-1β by qRT-PCR in AML12 cells transfected with Scram (control) or miR-34a mimics for 48 h. (G, H) Immunoblotting analysis of TRAF6, TLR4, CREBH, N-CREBH, and loading control β-actin protein in the AML12 cells transfected with Scram or miR-34a mimics for 48 h. Results represent mean ± standard deviation. The two-tailed student's t-test was used for statistical analyses of two-group comparisons. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus controls. Veh, vehicle; Scram, scramble miRNA; BSA, bovine serum albumin; PA, palmitic acid; TNFα, tumor necrosis factor alpha; qRT-PCR, real-time quantitative reverse transcription PCR; ApoB, apolipoprotein B; ApoE, apolipoprotein E; Mttp, microsomal triglyceride transfer protein; TG, triglyceride; GRP78, glucose regulatory protein 78; eIF2α-p, phosphor-eukaryotic initiation factor-2α; JNK-p, phosphor-c-Jun N-terminal kinase; Il-1β, interleukin-1β; TRAF6, tumor necrosis factor receptor-associated factor 6; TLR4, toll-like receptor 4; CREBH, cAMP-responsive element binding protein H; N-CREBH, N-terminal CREBH.

Figure 2

Increased miR-34a induced hepatocyte ballooning degeneration and fibrogenic status. AML12 cells were transfected with Scram (control) or miR-34a mimics for 48 h and subjected to the following analysis (A–D). (A) Hematoxylin–eosin staining and microscope imaging of the transfected AML12 cells. The arrows mark the ballooned AML12 cells. Scale bar, 20 μm. (B) mRNA expression of Shh by qRT-PCR. (C) qRT-PCR analysis of Tgfβ1 and Tgfβ2 mRNAs. (D) Immunoblotting analysis of TGFβ1 and TGFβ2 and loading control β-actin proteins. (E) mRNA expression of miR-34a by qRT-PCR in AML12 cells treated with Veh (H₂O) or Tgfβ2 (2 mg/mL) for 48 h. For cell treatment, two independent experiments were performed in triplicate. Results represent mean ± standard deviation. The two-tailed student's t-test was used for statistical analyses of two-group comparisons. ∗p < 0.05 and ∗∗p < 0.01 versus controls. qRT-PCR, real-time quantitative reverse transcription PCR; Shh, sonic hedgehog; TGF-β1/2, transforming growth factor-beta 1/2; Veh, vehicle; Scram, scramble miRNA.

Figure 3

miR-34a secreted from damaged hepatocytes mediated pathological communication between hepatocytes and HSCs. (A) qRT-PCR analysis of miR-34a contents in the conditional media of AML12 treated with Veh (bovine serum albumin) or PA (0.25 mM) for 18 h. (B) qRT-PCR analysis of miR-34a contents in the conditional media of AML12 transfected with Scram or miR-34a mimic for 48 h. (C, D) Conditional media from AML12 transfected with Scram or miR-34a for 48 h were used to treat HSCs for 48 h, followed by (C) immunoblotting analysis of GRP78, α-SMA, elF2α-p, TGFβ2, and loading control β-actin proteins in the treated HSCs. (D) qRT-PCR of Col1a1 and Grp78 mRNAs in the treated HSCs. (E) Immunoblotting analysis of α-SMA, TGFβ2, COL1A1, and loading control β-actin protein in the HSCs transfected with Scram or miR-34a for 48 h. (F) Conditional media from AML12 transfected with Scram or miR-34a for 48 h were used to treat wounded monolayer HSCs. Images were captured at 0-, 6-, 24-, and 30-h timepoints. The rate of wound healing is presented by the ratio (%) of wounded areas at each indicated time point to its initial wounded area at the 0-h timepoint. Results represent mean ± standard deviation. The two-tailed student's t-test was used for statistical analyses of two-group comparisons. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus controls. HSCs, hepatic stellate cells; qRT-PCR, real-time quantitative reverse transcription PCR; GRP78, glucose regulatory protein 78; eIF2α-p, phosphor-eukaryotic initiation factor-2α; JNK-p, phosphor-c-Jun N-terminal kinase; TGF-β2, transforming growth factor-beta 2; Veh, vehicle; Scram, scramble miRNA; PA, palmitic acid; α-SMA, alpha-smooth muscle actin; COL1A1, collagen type I alpha 1 chain.

Figure 4

Increased miR-34a in the livers of patients with steatosis, MASH, and liver fibrosis. (A) Representative histological images of liver tissues from patients with steatosis, MASH, and liver fibrosis by hematoxylin–eosin or Masson's trichrome staining. The arrows mark the ballooned hepatocytes, and the circle mark the inflammatory monocyte infiltration. Scale bar, 25 μm. (B, C) Quantitative reverse transcription PCR analysis of mRNA expression of ApoB, ApoE, Ldlr, and miR-34a in the livers of controls and patients with steatosis, MASH, and liver fibrosis. Control, n = 3; steatosis, n = 7; MASH, n = 9; liver fibrosis, n = 9. Results represent mean ± standard deviation. The two-tailed student's t-test was used for statistical analyses of two-group comparisons. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus controls. ApoB, apolipoprotein B; ApoE, apolipoprotein E; MASH, metabolic dysfunction-associated steatohepatitis; Ldlr, low-density lipoprotein receptor.

Figure 5

Recapitulation of pathological conditions of patients with MASLD and liver fibrosis in a human MASLD-like mouse model. Liver tissues from chow-fed controls or STAM mice (detailed in Materials and methods) at 6-, 8-, and 12-week-old were subjected to the following analysis. (A) TG and CHOL contents. (B, C) mRNA expression of Il-1β and Tnfα.(D) Immunoblotting analysis of α-SMA, COL1A1, and loading control β-actin proteins. (E) miRNA deep sequencing counts of miR-34a. (F) mRNA expression of miR-34a in primary hepatocytes (HCs) of control or STAM mice at 6- and 12-week-old. n = 5–10/group. Results represent mean ± standard deviation. The two-tailed student's t-test was used for statistical analyses of two-group comparisons. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus controls. MASLD, metabolic dysfunction-associated steatotic liver disease; TNFα, tumor necrosis factor alpha; Il-1β, interleukin-1β; α-SMA, alpha-smooth muscle actin; COL1A1, collagen type I alpha 1 chain; TG, triglyceride; CHOL, cholesterol.

Figure 6

Increased miR-34a mediates pathophysiological communication between hepatocytes and HSCs in liver fibrosis. Increased miR-34a induced by obesity and high-fat diet enhances hepatic de novo lipogenesis and disturbs biosynthesis of very-low density lipoprotein metabolism via down-regulation of Mttp, ApoB, and ApoE, leading to the accumulation of lipids in the liver (steatosis). Lipotoxicity derived from the accumulated lipids further induces ER stress and activates metabolic inflammation in the liver, driving the progressive liver injury to MASH, characterized by the activation of TNFα, IL-1β, and CREBH, hepatocyte ballooning, and fibrogenic genes. Simultaneously, secreted miR-34a from the damaged hepatocytes contributes to HSC activation and the subsequent induction of fibrogenic signaling molecules, such as α-SMA, Col1A1, and TGF-β, promoting ECM remodeling and the development of liver fibrosis. TNFα, tumor necrosis factor-alpha; ApoB, apolipoprotein B; ApoE, apolipoprotein E; Mttp, microsomal triglyceride transfer protein; Il-1β, interleukin-1β; CREBH, cAMP-responsive element binding protein H; TGF-β, transforming growth factor-beta; HSC, hepatic stellate cell; COL1A1, collagen type I alpha 1 chain; MASH, metabolic dysfunction-associated steatohepatitis; α-SMA, alpha-smooth muscle actin; ER, endoplasmic reticulum; ECM, extracellular matrix.