MicroRNA-9 limits hepatic fibrosis by suppressing the activation and proliferation of hepatic stellate cells by directly targeting MRP1/ABCC1
Retraction in: /10.3892/or.2023.8590
- Authors:
- Published online on: January 17, 2017 https://doi.org/10.3892/or.2017.5382
- Pages: 1698-1706
Abstract
Introduction
Chronic liver disease such as cirrhosis is a leading cause of death worldwide (1). Liver fibrosis, which is characterized by the excessive deposition of connective tissue in the extracellular matrix (ECM), is the final common endpoint for almost all chronic liver diseases (2). Therefore, numerous studies have focusd on identifying therapeutic targets for the treatment of hepatic fibrosis (3,4). Hepatic stellate cells (HSCs), which are located in the perisinusoidal Space of Disse, play a central role in the pathogenesis of liver fibrosis (5). Quiescent HSCs can be activated in response to chronic steatohepatitis (6). Activated HSCs stimulate ECM accumulation, such as α-smooth muscle actin (α-SMA) and college deposition, resulting in the occurrence of liver fibrosis (7). Therefore, an understanding of the proliferation and activation of HSCs is important for the development of effective antifibrotic therapies.
The family of ATP-binding cassette (ABC) proteins consists of important antitumor targets in pancreatic carcinoma (8). These proteins also play extremely important roles in liver physiology (9). Recently, high expression of several ABC transporters was described in hepatic progenitor cells (HPCs) in various liver diseases (10). The function of many ABC transporters has been extensively studied in hepatic steatosis (11). In addition, upregulated expression of ABC transporters, including multidrug resistance-associated protein 1 (MRP1/ABCC1), MRP3/ABCC3 and MRP4/ABCC4, has been demonstrated in activated HSCs (12). Although MRP1 has been reported to be required for the viability of activated HSCs, its presence and function in activated HSCs have scarcely been investigated.
Abnormal expression of microRNAs (miRNAs) has been found to be associated with human liver diseases, such as liver cancer, autoimmune liver disease and viral hepatitis (13–15). miRNAs are a class of short non-coding RNAs, ~19–22 nucleotides in length, that can bind to the 3′-untranslated regions (3′-UTRs) in target mRNA molecules, causing translation repression or the cleavage of target mRNAs (16). Previous research has shown that miRNAs can regulate the activation of HSCs (17), but little is known concerning the mechanisms of how miRNAs are involved in hepatic fibrogenesis.
Hedgehog (Hh), a fetal morphogenic signaling pathway protein, becomes activated in many types of chronic liver injury. The Hh ligands, including Sonic Hh, Indian Hh and Desert Hh, bind to the Hh receptor patched (Ptc) and release smoothened (Smo) into the cytoplasm. Cytoplasmic Smo translocates the glioblastoma (Gli) family proteins (Gli1, Gli2 and Gli3) into the nucleus, where they act as transcriptional activators of Hh signaling (18). Moreover, it has been reported that activation of the Hh signaling pathway promotes liver fibrosis (19). In the damaged liver, the Hh signaling pathway promotes activation of quiescent HSCs, which is associated with the accumulation of fibrous ECM in the liver (20).
In the present study, we investigated the involvement of miRNAs in the progression of liver fibrosis and discovered the abnormal expression of microRNA-9 (miR-9) and MRP1. Then, we focused on the effects of miR-9 on the proliferation and activation of HSCs. The signaling pathway involved in miR-9-mediated cell proliferation and activation of human HSCs was also explored.
Materials and methods
Human liver specimens
Fibrotic liver tissues (n=24) were obtained from patients clinically and pathologically diagnosed with liver fibrosis. Normal liver tissues (n=20) were obtained from the surgical resection of liver metastases. All samples were collected at the Affiliated Hospital of Shandong Medical College. The characteristics of the patients and healthy donors are listed in Table I. The protocol was approved by the Investigation and Ethics Committee of the Affiliated Hospital of Shandong Medical College. Each patient provided written informed consent. The collected tissue samples were divided into two portions; one was preserved in liquid nitrogen for the preparation of total RNA and total protein lysates, and the other was used for the isolation and culture of HSCs.
Cell culture
Primary human HSCs (pHSCs) were isolated from fragments of normal liver tissue obtained from the surgical resection of liver metastases, as described in detail elsewhere (21). pHSCs were cultured for three days, and then displayed a quiescent phenotype (qHSCs). After being cultured for 10 days, pHSCs transformed into activated cell types (aHSCs) (22). The obtained HSCs were cultured in RPMI medium supplemented with 20% fetal bovine serum (FBS), nonessential amino acids, 1.0 mM sodium pyruvate and antibiotic-antimycotic (all from Life Technologies, Carlsbad, CA, USA), as referred to a previous study (23).
LX-2 cells (human activated HSCs) were cultured with Dulbeccos modified Eagles medium (DMEM) (HyClone, Logan, UT, USA) containing 10% newborn calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
Quantitative real-time PCR
Total RNA was extracted from normal liver and liver cirrhosis tissues as well as qHSCs and aHSCs with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For mRNA quantification, 500 ng of RNA was used for the synthesis of cDNA with reverse transcriptase using the M-MLV First Strand kit (Taraka, Dalian, China) according to the manufacturer's instructions. cDNA (1 µl) was used for real-time PCR with GoTaq qPCR Master Mix (Promega, Madison, WI, USA). For each sample, the relative mRNA level was normalized to β-actin expression. The primers for qPCR are listed in Table II.
For miRNA quantification, the GoScript Reverse Transcription System kit (Promega) was used with the stem loop primer. For each sample, the relative mRNA level was normalized to U6. The specific forward primer for miR-9 was 5′-TCTTTGGTTATCTAGCTGTATGA-3′. Relative expression levels of miRNA or mRNA were analyzed using the Bio-Rad C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
Western blotting
Western blotting was performed as previously described (24). The primary antibodies used were rat anti-mouse MRP1, α-SMA, Col-1, Timp-1, Smo, Ptc, Gli2 and Gli3 (Abcam, Cambridge, UK). Secondary antibodies included horseradish peroxidase-conjugated goat anti-rat IgG (H+L) (Pierce, Rockford, IL, USA). The Western-Light chemiluminescent detection system (Image Station 4000 MM PRO, XLS180; Kodak, Rochester, NY, USA) was used to visualize the signals. The gray values were analyzed with BandScan5.0 software.
Transfection of miR-9 mimic and inhibitor
LX-2 cells were seeded 24 h prior to transfection into 24-well or 6-well plates or 6-cm dishes. LX-2 cells were transfected with an miRNA mimic control, miR-9 mimic, miRNA inhibitor control or miR-9 inhibitor (Applied Biosystems, Foster City, CA, USA) using Lipofectamine 2000 (Invitrogen) at a final concentration of 80 nM. The cells were harvested at 24 h (for RNA extraction) and at 48 h (for protein extraction).
Construction of the expression vector
For the expression plasmid construct, the wild-type MRP1 cDNA sequence without the 3′-UTR was selected and cloned into the pcDNA™3.2-DEST vector (Invitrogen).
Cell Counting Kit-8 (CCK-8) assay
Cell proliferation was measured using the CCK-8 kit (Solarbio, Beijing, China). In brief, 48 h after transfection with the miR-9 mimic or miR-9 inhibitor, LX-2 cells were seeded at 5×103 cells/well in 96-well plates in triplicate. At 0, 24, 48 and 72 h, 10 µl of CCK-8 solution mixed with 90 µl of DMEM was added to each well. After 2 h of incubation, absorbance was measured at 450 nm.
Luciferase reporter assay
The human MRP1 3′-UTR was amplified and cloned into the XbaI site of the pGL3-control vector (Promega), downstream of the luciferase gene, to generate the pGL3-MRP1-3′-UTR plasmid. As a control, the pGL3-MRP1-3′-UTR-mut plasmids were also constructed using cDNA fragments containing corresponding mutated nucleotides for miR-9. For the luciferase reporter assay, LX-2 cells were co-transfected with the luciferase reporter vectors and control and miR-9 mimics, control or miR-9 inhibitors, using Lipofectamine 2000. A β-actin promoter Renilla luciferase reporter was used for normalization. After 48 h, luciferase activity was analyzed using the Dual-Luciferase assay system (Promega), according to the manufacturer's protocols.
Experimental animal model
Male C57BL/6 mice (6-weeks-old) were purchased from the Experimental Animal Center of Shandong Province, housed with a 12-h light/dark cycle and allowed free access to normal food and water. To induce liver fibrosis, mice (n=10) received 0.6 ml/kg body weight CCl4 (Sigma-Aldrich, St. Louis, MO, USA) by i.p. injection, which was dissolved in corn oil, twice a week for eight weeks (25). Control mice (n=10) were injected i.p. with the same amount of saline. All mice were sacrificed to obtain serum and liver samples at 48 h post the last injection of CCl4 or corn oil. All experimental procedures were approved by the Institutional and Local Committee on the Care and Use of Animals of Shandong Medical College, and all animals received humane care according to the National Institutes of Health (Bethesda, MD, USA) guidelines.
To examine the effect of miR-9 in vivo, mice received 0.4 ml/kg body weight CCl4 dissolved in corn oil by i.p. injection, three times a week for three weeks (26). Next, the mice were randomly divided into four experimental groups: treatment with 0.4 ml/kg body weight of CCl4 twice a week in parallel with one i.p. injection of PBS (n=10, CCl4 group); miR-9 mimic (n=10, miR-9 group), Ad-con (n=10, Ad-emp group) or Ad-MRP1 (n=10, miR-9 + MRP1 group)/week for three weeks.
The liver histological sections from the control, CCl4 and miR-9-treated mice were examined by light microscopy after staining with hematoxylin and eosin (H&E).
Statistical analysis
All data are expressed as the mean ± SD of results derived from three independent experiments performed in triplicate. Statistical analysis was performed using Student's t-test and ANOVA. A difference was accepted as significant at p<0.05.
Results
Abnormal expression of miR-9 and MRP1 is observed in human fibrotic livers and cultured HSCs
qRT-PCR and western blotting were used to determine the expression of miR-9 and MRP1 in fibrotic liver tissues and cultured human HSCs. The results showed that the mRNA and protein levels of MRP1 were markedly increased in the human fibrotic liver tissues (Fig. 1B and C). A microRNA database was used to screen miRNA candidates targeted to MRP1. As a candidate target miRNA of MRP1, miR-9 showed significantly decreased expression in the human fibrotic liver tissues as compared with the healthy controls (Fig. 1A). Furthermore, qRT-PCR and western blotting results showed that MRP1 was upregulated in aHSCs compared with the qHSCs (Fig. 1E and F). The decreased level of miR-9 was also demonstrated in aHSCs (Fig. 1D). Thus, a decrease in miR-9 or an increase in MRP1 in fibrotic liver tissues plays an important role in the development of liver fibrosis.
Overexpression of miR-9 suppresses proliferation and activation of HSCs
In light of the decreased expression of miR-9 in activated HSCs, we next investigated the effect of miR-9 on the proliferation and activation of HSC cell line LX-2. miR-9 expression was markedly upregulated by miR-9 mimics (Fig. 2A). As determined by CCK-8 and BrdU assays, the transfection of miR-9 led to an inhibition of cell proliferation in the LX-2 cells as compared to that noted in the controls (Fig. 2B and C). In addition, we transfected miR-9 mimics into HSCs to clarify the role of miR-9 overexpression in HSC activation and collagen deposition. As shown in Fig. 2D and E, overexpression of miR-9 significantly suppressed mRNA and protein expression of α-SMA, Col-1 and Timp-1, which are characterized as genes involved in the activation of HSCs. In contrast, the mRNA expression and protein level of these genes were reversed by the miR-9 inhibitor, suggesting that miR-9 may suppress HSC activation as well as ECM deposition.
miR-9 directly targets MRP1/ABCC1
Examination of the homology showed that 12 nucleotides in the seed region of miR-9 were complementary to MRP1 bases (Fig. 3A). To determine whether miR-9 binds directly to the predicated sites of the MRP1 3′-UTR, we performed luciferase reporter assays. miR-9 significantly reduced MRP1 3′-UTR-dependent luciferase activity, but did not affect mutant reporter luciferase activity, whereas the miR-9 inhibitor had no effect on wild-type or mutant reporter luciferase activity (Fig. 3B). Western blot results showed that the protein expression of MRP1 was decreased in the miR-9-transfected HSCs, but increased in the miR-9 inhibitor-transfected HSCs (Fig. 3C). These results suggested the interaction between miR-9 and the 3′-UTR of MRP1.
Overexpression of MRP1 rescues the miR-9-mediated suppressive effect on the activation and proliferation of HSCs through the Hh pathway
To investigate whether the downregulation of MRP1 by miR-9 affects the expression of Hh ligands and Hh-target genes, we analyzed Smo, Ptc, Gli2 and Gli3 mRNA levels in HSCs by qRT-PCR. As compared to the control, mRNA expression of Smo, Ptc, Gli2 and Gli3 was significantly decreased after miR-9 transfection. However, these mRNA levels were increased after transfection with the miR-9 inhibitor (Fig. 4A). In addition, exogenous MRP1 was transfected into HSCs, and BrdU assay indicated that cell proliferation was decreased after miR-9 transfection, but increased after MRP1 transfection. However, the suppression of miR-9 on HSC proliferation was restored by the overexpression of MRP1 (Fig. 4B). Furthermore, miR-9 decreased the protein levels of Smo, Gli2 and α-SMA, but the overexpression of MRP1 significantly counteracted this inhibitory effect (Fig. 4C). Taken together, these results indicate that MRP1 overexpression rescues the miR-9-mediated suppressive effect on the activation and proliferation of HSCs by activating the Hh signaling pathway.
miR-9 limits hepatic fibrosis by suppressing expression of MRP1 in a CCl4-induced mouse hepatic fibrosis model in vivo
To better investigate the effect of miR-9 on liver fibrosis in vivo, we first examined the expression of miR-9 and MRP1 in fibrotic mouse liver tissues. qRT-PCR and western blotting demonstrated decreased miR-9 and increased MRP1 levels in the CCl4-induced liver fibrosis mouse model (Fig. 5A). Western blotting suggested that the protein expression of Timp-1, α-SMA and Col-1 was increased in the CCl4 group. miR-9 reduced the expression of Timp-1, α-SMA and Col-1 in the CCl4-induced mouse model, whereas these effects were restored by overexpression of MRP1 (Fig. 5B). H&E staining showed that liver tissues in the control group exhibited normal structure with no abnormal morphological changes. In contrast, the number of fibrotic septa was increased in the livers of animals from the CCl4 group, and the normal lobular architecture was lost. The group transfected with miR-9 showed less pathological damage than that observed in the CCl4 group. Furthermore, the results showed that the overexpression of MRP1 suppressed the effects of miR-9 on CCl4-induced liver fibrosis (Fig. 5C). These results indicate that miR-9 treatment slowed the progression of liver fibrosis in CCl4-induced mice, resulting in a reduction in the deposition of ECM in the liver by reducing MRP1.
Discussion
Liver fibrosis represents a huge burden to health care services worldwide. The establishment of HSCs as primary effector cells for the deposition of ECM in normal and fibrotic liver was a milestone discovery in understanding the pathogenesis of hepatic fibrosis (27). Since then, cellular signaling molecules, cell membrane receptors and transcription factors in HSCs have been widely investigated and found to promote hepatic fibrogenesis (5,28). However, the factors and signaling cascades that prevent the pathological process of liver fibrosis are poorly understood. In addition, it has been demonstrated that multidrug resistance-associated protein 1 (MRP1) contributes to HSC activation in the process of liver fibrosis (12). Recent studies have reported that expression of dysregulated miRNAs exerts an anti-fibrotic effect in mouse models of CCl4-induced liver fibrosis (14,24). Our results showed that the protein and mRNA expression of MRP1 was increased in liver cirrhosis patients. The expression of miRNA-9 (miR-9), a candidate target miRNA of MRP1, was decreased in patients with liver cirrhosis. Thus, we mainly focused on the effects of miR-9 on HSC proliferation and activation as well as the underlying molecular mechanism.
The effects of various miRNAs on liver fibrosis have been illustrated in recent studies. The miRNAs involved in hepatic fibrosis can be broadly categorized as pro-fibrotic or anti-fibrotic miRNAs: pro-fibrotic miRNAs are upregulated and anti-fibrotic miRNAs are downregulated during fibrogenesis. For example, miR-29 is an anti-fibrogenic miRNA and it has been reported to play a crucial role in alleviating CCl4-induced liver fibrosis (29). In contrast, miR-34a has been reported to promote HSC activation (30). A previous study reported that miR-9 was downregulated in activated HSCs (31). In the present study, the expression of miR-9 was downregulated in liver cirrhosis patient tissues. Moreover, miR-9 plays a role in the treatment of cardiac fibrosis. It has been found that miR-9 regulates cardiac fibroblast proliferation and collagen production (32). The present study found that transfection of miR-9 led to an inhibition of cell proliferation in human HSCs (LX-2) as compared to that noted in the control cells. In response to liver injury, quiescent HSCs are activated and develop a myofibroblast-like phenotype that expresses smooth muscle actin (α-SMA) and profibrogenic genes, such as collagen type I (Col 1) and tissue inhibitor of metal protease-1 (Timp-1) (24). It has been reported that the expression of α-SMA and Col-1 was inhibited by Xia-yu-xue decoction (XYXD), and HSC activation was suppressed in CCl4-induced liver fibrosis in vivo (33). In addition, human LX-2 cells also secrete TIMP-1, which is involved in HSC activation (34). In the present study, miR-9 inhibited the expression of pro-fibrotic genes such as α-SMA, Col-1 and TIMP-1 in LX-2 cells, suggesting that miR-9 prevents the activation of HSCs. These findings imply that miR-9 may negatively regulate liver fibrogenesis by inhibiting HSC activation and collagen synthesis in HSCs.
High expression of MRP1, an ABC transporter, is associated with enhanced resistance to cell death (9). MRP1 has been reported to be required for the viability of activated HSCs (12). In the present study, cirrhotic liver tissues showed upregulated levels of MRP1 and downregulated levels of miR-9. Moreover, Targetscan predicted that MRP1 is a target gene of miR-9 in LX-2 cells. Our findings suggest that miR-9 regulates the expression of MRP1 at both the mRNA and protein levels by binding directly to the 3′-UTR of MRP1. Thus, we may conclude that miR-9 mediates the proliferation and activation of HSCs, and downregulates MRP1 expression by targeting MRP1 directly in LX-2 cells.
Emerging evidence shows that the Hedgehog (Hh) pathway critically regulates the growth and repair responses in the liver (35). The level of Hh pathway activation appears to be proportional to the severity and duration of liver injury in both rodents and humans (36). Investigation of the mechanisms underlying Hh ligand production indicates that Hh signaling may likely be activated when major liver reconstruction is required (37). It has been demonstrated that overexpression of several key molecules in the Hh pathway (Smo and Ptc) accompanied the upregulation of the α-SMA level, which is a marker of activated HSCs in rat fibrotic liver (38). These findings provide a rationale for inhibiting the activation of the Hh pathway in the context of treatment for liver fibrosis. Herein, we showed that miR-9 decreased the expression of Smo, Ptc, Gli2 and Gli3, while the miR-9 inhibitor increased these expression levels in HSCs. Moreover, the miR-9-mediated suppression of cell proliferation and activation of HSCs was attenuated by MRP1 transfection. A previous study showed that inhibition of Hh signaling could restrain MRP1 expression in cancer (39). These data consistently revealed that miR-9 disrupted Hh signaling by targeting MRP1 in activated HSCs. To better investigate the function of miR-9 in liver fibrosis in vivo, we analyzed the expression of ECM protein and the morphological changes in liver tissues from C57BL/6 mice exposed to CCl4. The results indicated that miR-9 treatment reduced the progression of liver fibrosis in the CCl4-induced liver fibrosis mouse model, resulted in reduced MRP1 levels and a subsequent reduction in the deposition of ECM in the liver.
Taken together, the present study demonstrated that miR-9 suppresses the proliferation and activation of HSCs by targeting MRP1 and also inhibits the activation of Hh to prevent liver fibrosis by decreasing the expression of Hh target genes, Smo, Ptc, Gli2 and Gli3. These findings indicate that miR-9 has great potential to be used as a therapeutic agent for the treatment of liver fibrosis.
References
|
Kim Y, Ejaz A, Tayal A, Spolverato G, Bridges JF, Anders RA and Pawlik TM: Temporal trends in population-based death rates associated with chronic liver disease and liver cancer in the United States over the last 30 years. Cancer. 120:3058–3065. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Popov Y and Schuppan D: Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies. Hepatology. 50:1294–1306. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Fagone P, Mangano K, Pesce A, Portale TR, Puleo S and Nicoletti F: Emerging therapeutic targets for the treatment of hepatic fibrosis. Drug Discov Today. 21:369–375. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Fagone P, Mangano K, Mammana S, Pesce A, Pesce A, Caltabiano R, Giorlandino A, Portale TR, Cavalli E, Lombardo GA, et al: Identification of novel targets for the diagnosis and treatment of liver fibrosis. Int J Mol Med. 36:747–752. 2015.PubMed/NCBI | |
|
Puche JE, Saiman Y and Friedman SL: Hepatic stellate cells and liver fibrosis. Compr Physiol. 3:1473–1492. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Lade A, Noon LA and Friedman SL: Contributions of metabolic dysregulation and inflammation to nonalcoholic steatohepatitis, hepatic fibrosis, and cancer. Curr Opin Oncol. 26:100–107. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Peverill W, Powell LW and Skoien R: Evolving concepts in the pathogenesis of NASH: Beyond steatosis and inflammation. Int J Mol Sci. 15:8591–8638. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
König J, Hartel M, Nies AT, Martignoni ME, Guo J, Büchler MW, Friess H and Keppler D: Expression and localization of human multidrug resistance protein (ABCC) family members in pancreatic carcinoma. Int J Cancer. 115:359–367. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Borst P and Elferink RO: Mammalian ABC transporters in health and disease. Annu Rev Biochem. 71:537–592. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Ros JE, Roskams TA, Geuken M, Havinga R, Splinter PL, Petersen BE, LaRusso NF, van der Kolk DM, Kuipers F, Faber KN, et al: ATP binding cassette transporter gene expression in rat liver progenitor cells. Gut. 52:1060–1067. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Jeon BH, Lee YH, Yun MR, Kim SH, Lee BW, Kang ES, Lee HC and Cha BS: Increased expression of ATP-binding cassette transporter A1 (ABCA1) as a possible mechanism for the protective effect of cilostazol against hepatic steatosis. Metabolism. 64:1444–1453. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Hannivoort RA, Dunning S, Borght S Vander, Schroyen B, Woudenberg J, Oakley F, Buist-Homan M, van den Heuvel FA, Geuken M, Geerts A, et al: Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells. Hepatology. 48:624–634. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Hayes CN and Chayama K: MicroRNAs as biomarkers for liver disease and hepatocellular carcinoma. Int J Mol Sci. 17:2802016. View Article : Google Scholar : PubMed/NCBI | |
|
Kitano M and Bloomston PM: Hepatic stellate cells and microRNAs in pathogenesis of liver fibrosis. J Clin Med. 5:52016. View Article : Google Scholar : | |
|
Szabo G and Bala S: MicroRNAs in liver disease. Nat Rev Gastroenterol Hepatol. 10:542–552. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Bartel DP: MicroRNAs: Target recognition and regulatory functions. Cell. 136:215–233. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Li G, Li J, Li C, Qi H, Dong P, Zheng J and Yu F: MicroRNA-125a-5p contributes to hepatic stellate cell activation through targeting FIH1. Cell Physiol Biochem. 38:1544–1552. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Omenetti A, Choi S, Michelotti G and Diehl AM: Hedgehog signaling in the liver. J Hepatol. 54:366–373. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Choi SS, Omenetti A, Syn WK and Diehl AM: The role of Hedgehog signaling in fibrogenic liver repair. Int J Biochem Cell Biol. 43:238–244. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Chen Y, Choi SS, Michelotti GA, Chan IS, Swiderska-Syn M, Karaca GF, Xie G, Moylan CA, Garibaldi F, Premont R, et al: Hedgehog controls hepatic stellate cell fate by regulating metabolism. Gastroenterology. 143:1319–1329.e1-11. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Rombouts K and Carloni V: Determination and characterization of tetraspanin-associated phosphoinositide-4 kinases in primary and neoplastic liver cells. Methods Mol Biol. 1376:203–212. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Görbig MN, Ginès P, Bataller R, Nicolás JM, Garcia-Ramallo E, Cejudo P, Sancho-Bru P, Jiménez W, Arroyo V and Rodés J: Human hepatic stellate cells secrete adrenomedullin: Potential autocrine factor in the regulation of cell contractility. J Hepatol. 34:222–229. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Jalan R, De Chiara F, Balasubramaniyan V, Andreola F, Khetan V, Malago M, Pinzani M, Mookerjee RP and Rombouts K: Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension. J Hepatol. 64:823–833. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Hyun J, Wang S, Kim J, Rao KM, Park SY, Chung I, Ha CS, Kim SW, Yun YH and Jung Y: MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression. Nat Commun. 7:109932016. View Article : Google Scholar : PubMed/NCBI | |
|
Roderburg C, Urban GW, Bettermann K, Vucur M, Zimmermann H, Schmidt S, Janssen J, Koppe C, Knolle P, Castoldi M, et al: Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology. 53:209–218. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang DW, Zhao YX, Wei D, Li YL, Zhang Y, Wu J, Xu J, Chen C, Tang H, Zhang W, et al: HAb18G/CD147 promotes activation of hepatic stellate cells and is a target for antibody therapy of liver fibrosis. J Hepatol. 57:1283–1291. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Friedman SL: Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 328:1828–1835. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Abu-Elsaad NM and Elkashef WF: Modified citrus pectin stops progression of liver fibrosis by inhibiting galectin-3 and inducing apoptosis of stellate cells. Can J Physiol Pharmacol. 94:554–562. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Gao B, Chang C, Zhou J, Zhao T, Wang C, Li C and Gao G: Pycnogenol protects against rotenone-induced neurotoxicity in PC12 cells through regulating NF-κB-iNOS signaling pathway. DNA Cell Biol. 34:643–649. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Yan G, Li B, Xin X, Xu M, Ji G and Yu H: MicroRNA-34a promotes hepatic stellate cell activation via targeting ACSL1. Med Sci Monit. 21:3008–3015. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Ji J, Zhang J, Huang G, Qian J, Wang X and Mei S: Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation. FEBS Lett. 583:759–766. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Wang L, Ma L, Fan H, Yang Z, Li L and Wang H: MicroRNA-9 regulates cardiac fibrosis by targeting PDGFR-β in rats. J Physiol Biochem. 72:213–223. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Liu C, Yuan X, Tao L, Cheng Z, Dai X, Sheng X and Xue D: Xia-yu-xue decoction (XYXD) reduces carbon tetrachloride (CCl4)-induced liver fibrosis through inhibition hepatic stellate cell activation by targeting NF-κB and TGF-β1 signaling pathways. BMC Complement Altern Med. 15:2012015. View Article : Google Scholar : PubMed/NCBI | |
|
Xu L, Hui AY, Albanis E, Arthur MJ, O'Byrne SM, Blaner WS, Mukherjee P, Friedman SL and Eng FJ: Human hepatic stellate cell lines, LX-1 and LX-2: New tools for analysis of hepatic fibrosis. Gut. 54:142–151. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Omenetti A, Popov Y, Jung Y, Choi SS, Witek RP, Yang L, Brown KD, Schuppan D and Diehl AM: The hedgehog pathway regulates remodelling responses to biliary obstruction in rats. Gut. 57:1275–1282. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Fleig SV, Choi SS, Yang L, Jung Y, Omenetti A, VanDongen HM, Huang J, Sicklick JK and Diehl AM: Hepatic accumulation of Hedgehog-reactive progenitors increases with severity of fatty liver damage in mice. Lab Invest. 87:1227–1239. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Choi SS, Omenetti A, Witek RP, Moylan CA, Syn WK, Jung Y, Yang L, Sudan DL, Sicklick JK, Michelotti GA, et al: Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis. Am J Physiol Gastrointest Liver Physiol. 297:G1093–G1106. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Lian N, Jiang Y, Zhang F, Jin H, Lu C, Wu X, Lu Y and Zheng S: Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells. Lab Invest. 95:790–803. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang Y, Laterra J and Pomper MG: Hedgehog pathway inhibitor HhAntag691 is a potent inhibitor of ABCG2/BCRP and ABCB1/Pgp. Neoplasia. 11:96–101. 2009. View Article : Google Scholar : PubMed/NCBI |
