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Microrna‑200A Suppresses Epithelial‑To‑Mesenchymal Transition in Rat Hepatic Stellate Cells Via GLI Family Zinc Finger 2

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Microrna‑200A Suppresses Epithelial‑To‑Mesenchymal Transition in Rat Hepatic Stellate Cells Via GLI Family Zinc Finger 2 MOLECULAR MEDICINE REPORTS 12: 8121-8128, 2015 MicroRNA‑200a suppresses epithelial‑to‑mesenchymal transition in rat hepatic stellate cells via GLI family zinc finger 2 FUJUN YU1, YIHU ZHENG2, WEILONG HONG3, BICHENG CHEN3, PEIHONG DONG1 and JIANJIAN ZHENG3 Departments of 1Infectious Diseases and 2General Surgery; 3Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China Received December 31, 2014; Accepted September 25, 2015 DOI: 10.3892/mmr.2015.4452 Abstract. Hepatic stellate cells (HSCs) have an important Introduction role in liver fibrosis. Epithelial-to-mesenchymal transition (EMT), which is promoted by the Hedgehog (Hh) signaling Liver fibrosis, characterized by excess production and depo- pathway, is involved in the activation of HSCs. MicroRNAs sition of extracellular matrix (ECM) along with loss of liver (miRNAs/miRs) have been reported to be involved in the function and disruption of liver structure, is a wound-healing progression of liver fibrosis. A previous study indicated response to chronic liver injury (1,2). It is well known that that the activation of HSCs was suppressed by miR-200a hepatic stellate cells (HSCs) have an important role in liver via targeting transforming growth factor-β2 and β-catenin. fibrosis. The activation and proliferation of resident hepatic stel- However, whether miR-200a is able to regulate the EMT in late cells (HSCs) has been considered as a central event in the HSCs has remained elusive. The present study revealed that progression of liver fibrosis. During fibrosis progression, quies- miR-200a was decreased in vitro and in vivo during liver cent (Q)-HSCs become activated and transdifferentiate into fibrosis. Furthermore, miR-200a overexpression resulted myofibroblastic HSCs (MF‑HSCs) that express smooth‑muscle in the inhibition of proliferation, α-SMA expression and α-actin (α‑SMA) as a marker (3). Activation of HSC, the major extracellular matrix production of activated HSCs. Of note, cell type promoting synthesis and deposition of ECM proteins, miR‑200a overexpression reduced myofibroblastic markers, results in an imbalance between ECM protein generation including α-SMA, type I collagen and desmin, and increased and their degradation in the liver (4). Therefore, a possible the epithelial cell marker E‑cadherin. These results were therapeutic strategy for treating liver fibrosis is to inhibit the further confirmed by immunofluorescence staining. Further activation of HSCs. study showed that the expression of genes associated with Hh Epithelial-to-mesenchymal transition (EMT) is the process signaling, including Hhip, Shh and Gli1, were not affected by by which epithelial cells gradually lose their epithelial signa- miR-200a. However, Gli2, a downstream signaling protein of tures while acquiring the characteristics of mesenchymal the Hh pathway, was inhibited by miR‑200a and confirmed cells (5). A growing body of evidence implied that EMT has as a target of miR-200a using a dual luciferase reporter assay. an important role in liver fibrosis (6,7). In particular, it has In addition, the inhibition of the Hh pathway by miR-200a been confirmed that myofibroblasts can be supplemented resulted in an increase of BMP-7 and Id2 as well as a reduction from hepatocytes by EMT during hepatic fibrosis (8). EMT of Snai1 and S100A4. Collectively, the results of the present has emerged as a promising therapeutic target for the attenu- study demonstrated that miR-200a suppressed the EMT ation of liver fibrosis. Hedgehog (Hh) signaling has been process in HSCs, at least in part, via Gli2. identified to be involved in EMT. Choi et al (9) demonstrated that EMT is regulated by Hh signaling during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis. Omenetti et al (7) found that EMT responses to bile duct liga- Correspondence to: Mr. Jianjian Zheng, Key Laboratory of tion were enhanced in patched‑deficient mice, which display Surgery, The First Affiliated Hospital of Wenzhou Medical excessive activation of the Hh pathway. A previous study University, 2 Fuxue Lane, Wenzhou, Zhejiang 325000, P.R. China showed that leptin, a pro-EMT factor, activates Hh signaling E-mail: [email protected] to alter the expression of genes which control cell fate and have important implications in liver fibrosis (10). These findings Dr Peihong Dong, Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, 2 Fuxue Lane, suggested that Hh signaling contributes to EMT. Wenzhou, Zhejiang 325000, P.R. China MicroRNAs (miRNAs) are endogenous small (18-22 nt) E-mail: [email protected] non-coding RNAs that regulate the expression of proteins involved in diverse cellular and developmental processes, Key words: microRNA-200a, epithelial-to-mesenchymal transition, including differentiation, apoptosis and oncogenesis (11). hepatic stellate cells, hedgehog pathway, GLI family zinc finger 2 miRNAs specifically bind to the 3'‑untranslated region (3'‑UTR) of their respective target mRNAs to decrease their stability and translation to regulate protein expression (12). 8122 YU et al: miR-200A IN HEPATIC STELLATE CELLS During liver fibrosis, miRNAs contribute to the activa- (>98%) was confirmed by immunocytochemical staining for tion status of HSCs and function as HSC regulators in liver α-SMA. The present study was approved by the University fibrosis. Sun et al (13) reported that miR‑200a overexpression Animal Care and Use Committee of the Wenzhou Medical suppressed HSC activation and proliferation by targeting University (Wenzhou, China). TGF-β2 and β-catenin. Given the complexity of the EMT, it is likely that miRNAs regulate most genes involved in the miRNA transfection. Cells were seeded in a six-well plate at a EMT. Previously, it has been reported that the miR-200 family density of 1x106 cells per well. The following day, medium was regulates transforming growth factor (TGF)-β1-induced renal replaced with Opti-Minimum Essential Medium (Invitrogen; tubular EMT through the Smad pathway by targeting the Thermo Fisher Scientific) and cells were transiently trans- expression of zinc finger E-box binding homeobox 1 (ZEB1) fected with miR‑200a mimics (60 nM) or the miR‑negative and ZEB2 (14). However, it has remained elusive whether control (NC; 60 nM; GenePharma, Shanghai, China) using miR-200a regulates EMT in HSCs. Lipofectamine 2000 (Invitrogen) for 48 h in all experiments. The present study assessed the effects of miR-200a on the proliferation of activated HSCs; furthermore, western blot Rat model of CCl4‑induced liver injury. Male Sprague-Dawley analysis, reverse-transcription quantitative polymerase chain rats (n=20; weighy, 180‑220 g; age, 7 weeks) were obtained reaction (RT-qPCR) and immunofluorescence were used to from the Experimental Animal Center of the Wenzhou assess the expression of epithelial marker E‑cadherin and Medical University, and housed in an environmentally myofibroblastic markers α-SMA, type I collagen and desmin controlled room (23±2˚C; 55±10% humidity) with a 12 h light/ in order to investigate the effects of miR-200a on the EMT dark cycle, and were provided ad libitum access to food and of cultured HSCs. In addition, the effects of miR-200a on water. Liver fibrosis was generated by a 6 week treatment with the expression of Hh pathway-associated genes, including CCl4 [CCl4/olive oil, 1:1 (v/v) per kg body weight by intra- Hh-interacting protein (Hhip), Sonic Hh (Shh) and GLI family peritoneal injection twice weekly] for six weeks as previously zinc finger 1 (Gli1) and Gli2, as well as genes associated with described (16). Twenty rats were randomly divided into two the EMT, including bone morphogenetic protein-7 (BMP-7), groups. Rats in group 1 (n=10) received injections with olive oil inhibitor of DNA binding 2 (Id2), Snail family zinc finger 1 (vehicle control) and rats in group 2 (n=10) received injections (Snai1) and S100 calcium-binding protein A4 (S100A4) were of CCl4 twice weekly. The animal experimental protocol was assessed. A luciferase reporter assay was then employed to approved by the University Animal Care and Use Committee investigate whether Gli2 was a direct target of miR-200a. The (Wenzhou Medical University, Wenzhou, China). Rats were present study suggested that miR-200a suppressed EMT in rat sacrificed under anesthesia byintraperitoneal injection of 10% HSCs, at least in part, via Gli2. chloral hydrate (Sigma‑Aldrich; 4 ml/kg body weight) after the last CCl4 injection and liver tissues were harvested for Materials and methods further analysis. Materials. CCl4 was obtained from Sigma-Aldrich (St. Immunofluorescence microscopy. Primary HSCs on day 3 Louis, MO, USA) and antibodies, including rabbit poly- following inoculation were plated on 18-mm cover glasses clonal anti‑type I collagen (cat. no. ab34710), anti‑desmin in DMEM at a density of 1x105 cells/well and incubated for (cat. no. ab15200) and anti-S100A4 (cat. no. ab27957), and mouse 24 h. The cells were then transfected with miR-200a mimics monoclonal anti‑E‑cadherin (cat. no. ab76055), anti‑α-SMA or miR-NC for 48 h. Following washing with phosphate-buff- (cat. no. ab7817), and anti-β‑actin (cat. no. ab6276) were ered saline (PBS; Wenzhou Changfeng Biotechnology Co., obtained from Abcam (Cambridge, MA, USA). Antibodies Ltd., Wenzhou, China) and fixing in acetic acid/ethanol including goat polyclonal anti-BMP-7 (cat. no. sc‑9305), (30% ethanol and 10% acetic acid; Wenzhou Changfeng anti-Id2 (cat. no. sc‑26328), and anti‑Hhip (cat. no. sc‑9406), Biotechnology Co., Ltd.) for 5 min at ‑20˚C, non‑specific and rabbit polyclonal anti-Snai1 (cat. no. sc-28199), anti-Shh binding was blocked with 5% goat serum (Sigma‑Aldrich) (cat. no. sc-9024), anti-Gli1 (cat. no. sc‑20687) and anti‑Gli2 in PBS for 1 h at room temperature and the cells were then (cat. no. sc‑28674) were purchased from Santa Cruz incubated overnight at 4˚C with primary antibodies against Biotechnology (Dallas, TX, USA).
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