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MiR?375 affects the hedgehog signaling pathway by downregulating RAC1 to inhibit hepatic stellate cell viability and epithelial?mesenchymal transition.

ABSTRACT: MicroRNAs (miRNAs/miRs) are a class of non?coding RNAs that serve crucial roles in liver cancer and other liver injury diseases. However, the expression profile and mechanisms underlying miRNAs in liver fibrosis are not completely understood. The present study identified the novel miR?375/Rac family small GTPase 1 (RAC1) regulatory axis in liver fibrosis. Reverse transcription?quantitative PCR was performed to detect miR?375 expression levels. MTT, flow cytometry and western blotting were performed to explore the in vitro roles of miR?375. The dual?luciferase reporter gene assay was performed to determine the potential mechanism underlying miR?375 in liver fibrosis. miR?375 expression was significantly downregulated in liver fibrosis tissues and cells compared with healthy control tissues and hepatocytes, respectively. Compared with the pre?negative control group, miR?375 overexpression inhibited mouse hepatic stellate cell (HSC) viability and epithelial?mesenchymal transition, and alleviated liver fibrosis. The dual?luciferase reporter assay results demonstrated that miR?375 bound to RAC1. Moreover, the results indicated that miR?375 regulated the hedgehog signaling pathway via RAC1 to restrain HSC viability and EMT, thus exerting its anti?liver fibrosis function. The present study identified the miR?375/RAC1 axis as a novel regulatory axis associated with the development of liver fibrosis.

SUBMITTER: Liang Z

PROVIDER: S-EPMC7809903 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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miR‑375 affects the hedgehog signaling pathway by downregulating RAC1 to inhibit hepatic stellate cell viability and epithelial‑mesenchymal transition.

Liang Zhiwei Z Li Jian J Zhao Longshuan L Deng Yilei Y

Molecular medicine reports 20210105 3

MicroRNAs (miRNAs/miRs) are a class of non‑coding RNAs that serve crucial roles in liver cancer and other liver injury diseases. However, the expression profile and mechanisms underlying miRNAs in liver fibrosis are not completely understood. The present study identified the novel miR‑375/Rac family small GTPase 1 (RAC1) regulatory axis in liver fibrosis. Reverse transcription‑quantitative PCR was performed to detect miR‑375 expression levels. MTT, flow cytometry and western blotting were perfor ...[more]

PMID: 33398380

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Project description:The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism.Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1? (HIF1?) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity.Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1?. Inhibitors of Hh signaling, HIF1?, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh.Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Project description:Background & aimsCirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP.MethodsPrimary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis.ResultsExpression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression.ConclusionsGlutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

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MiR?375 affects the hedgehog signaling pathway by downregulating RAC1 to inhibit hepatic stellate cell viability and epithelial?mesenchymal transition.

Publications

miR‑375 affects the hedgehog signaling pathway by downregulating RAC1 to inhibit hepatic stellate cell viability and epithelial‑mesenchymal transition.

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