Yuyan Han

Functional analysis of microRNAs in human hepatocellular cancer stem cells

MicroRNAs are endogenous small non‐coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self‐renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL‐6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL‐6 and Twist. We demonstrated that conserved let‐7 and miR‐181 family members were up‐regulated in HSCs by global microarray‐based microRNA profiling followed by validation with real‐time polymerase chain reaction. Importantly, inhibition of let‐7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR‐181 led to a reduction in HSCs motility and invasion. Knocking down IL‐6 and Twist in HSCs significantly reduced let‐7 and miR‐181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let‐7 directly targets SOCS‐1 and caspase‐3, whereas miR‐181 directly targets RASSF1A, TIMP3 as well as nemo‐like kinase (NLK). In conclusion, alterations of IL‐6‐ and Twist‐regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let‐7/miR‐181s suggesting that let‐7 and miR‐181 may be molecular targets for eradication of hepatocellular malignancies.

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.

Role of stem cell factor and granulocyte colony-stimulating factor in remodeling during liver regeneration†‡

Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte macrophage colony‐stimulating factors (GM‐CSFs), and stem cell factors (SCFs) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblotting and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real‐time polymerase chain reaction (PCR). There was a significant, stable increase in SCF and GM‐CSF levels until 7 days after PH. Real‐time PCR analysis revealed significant increases of key remodeling molecules, such as S100 calcium‐binding protein A4 (S100A4) and miR‐181b, after SCF plus GM‐CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCFs and GM‐CSFs in response to transforming growth factor‐beta (TGF‐β). When SMCCs were incubated with TGF‐β plus anti‐SCF+GM‐CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF+GM‐CSF significantly increased matrix metalloproteinases (MMP‐2 and MMP‐9) messenger RNA as well as miR‐181b expression, along with a reduction of metalloproteinase inhibitor 3. Levels of MMP‐2, MMP‐9, and miR‐181b were also up‐regulated in rat liver and isolated cholangiocytes after PH. Conclusion: Our data suggest that altered expression of SCF+GM‐CSF after PH can contribute to biliary remodeling (e.g., post‐transplantation) by functional deregulation of the activity of key signaling intermediates involved in cell expansion and multipotent differentiation. (HEPATOLOGY 2012;;55:209–221)

Regulation of the Extrinsic Apoptotic Pathway by MicroRNA-21 in Alcoholic Liver Injury

Background: miR-21 is an anti-apoptotic microRNA, and its role in alcoholic liver disease (ALD) is unknown. Results: miR-21, increased in ALD and regulated by IL-6/Stat3, is essential for transformation, survival, and liver fibrosis. Conclusion: miR-21 plays a protective role against alcoholic hepatitis through the anti-extrinsic apoptotic pathway. Significance: Understanding the recovery function of miR-21 may have important implications for patients with ALD. IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5′-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.

The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis†‡

Cholangiocarcinoma (CCA) is a biliary cancer arising from damaged bile ducts. Epithelial‐mesenchymal transition (EMT) occurs as epithelial cells begin to resemble mesenchymal cells leading to increased invasion potential as the extracellular matrix (ECM) degrades. Histamine exerts its effects by way of four receptors (H1‐H4 HRs). Clobenpropit, a potent H4HR agonist, inhibits mammary adenocarcinoma growth. We have shown that (1) cholangiocytes and CCA cells express H1‐H4 HRs and (2) the H3HR decreases CCA proliferation. We evaluated the effects of clobenpropit on CCA proliferation, invasion, EMT phenotypes, and ECM degradation. In vitro, we used CCA cell lines to study proliferation, signaling pathways, and the morphological invasive potential. Gene and protein expression of the hepatobiliary epithelial markers CK‐7, CK‐8, and CK‐19, the focal contact protein paxillin, and the mesenchymal markers fibronectin, s100A4, and vimentin were evaluated. Cell invasion across an ECM layer was quantitated and matrix metalloproteinase‐1, ‐2, ‐3, ‐9, and ‐11 gene and protein expression was examined. Evaluation of the specific role of H4HR was performed by genetic knockdown of the H3HR and overexpression of H4HR. Proliferation was evaluated by proliferating cellular nuclear antigen immunoblotting. In vivo, xenograft tumors were treated with either vehicle or clobenpropit for 39 days. Tumor volume was recorded every other day. Clobenpropit significantly decreased CCA proliferation by way of a Ca2+‐dependent pathway and altered morphological development and invasion. Loss of H3HR expression or overexpression of H4HR significantly decreased CCA proliferation. In vivo, clobenpropit inhibited xenograft tumor growth compared with controls. Conclusion: Modulation of H4HR by clobenpropit disrupts EMT processes, ECM breakdown, and invasion potential and decreases tumor growth. Interruption of tumorigenesis and invasion by histamine may add to therapeutic advances for CCAs. (HEPATOLOGY 2011;)

Secretin Stimulates Biliary Cell Proliferation by Regulating Expression of MicroRNA 125b and MicroRNA let7a in Mice

BACKGROUND & AIMS Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. METHODS Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. RESULTS Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. CONCLUSIONS After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin → melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.

Recent advances in the morphological and functional heterogeneity of the biliary epithelium

This review focuses on the recent advances related to the heterogeneity of different-sized bile ducts with regard to the morphological and phenotypical characteristics, and the differential secretory, apoptotic and proliferative responses of small and large cholangiocytes to gastrointestinal hormones/peptides, neuropeptides and toxins. We describe several in vivo and in vitro models used for evaluating biliary heterogeneity. Subsequently, we discuss the heterogeneous proliferative and apoptotic responses of small and large cholangiocytes to liver injury and the mechanisms regulating the differentiation of small into large (more differentiated) cholangiocytes. Following a discussion on the heterogeneity of stem/progenitor cells in the biliary epithelium, we outline the heterogeneity of bile ducts in human cholangiopathies. After a summary section, we discuss the future perspectives that will further advance the field of the functional heterogeneity of the biliary epithelium.

Activation of alpha1-adrenergic receptors stimulate the growth of small mouse cholangiocytes Via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1†‡

Small cholangiocytes proliferate via activation of calcium (Ca2+)‐dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate–dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca2+‐dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α1‐adrenergic agonist stimulating intracellular D‐myo‐inositol‐1,4,5‐triphosphate (IP3)/Ca2+ levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP3/Ca2+‐dependent signaling. Phenylephrine stimulated the production of intracellular IP3. The Ca2+‐dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca2+‐dependent DNA‐binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine‐induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca2+‐dependent activation of NFAT2 and Sp1. Conclusion: Selective stimulation of Ca2+‐dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins. (HEPATOLOGY 2010;53:628‐639)

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats.

Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3′,5′‐monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl−/HCO  3− AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin‐stimulated choleresis in cholestatic bile‐duct–ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down‐regulation of cAMP‐dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N‐acetyltransferase (AANAT; the rate‐limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo‐Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl−/HCO  3− AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl−/HCO  3− AE2 and ablated secretin‐stimulated biliary secretion in these cells. Conclusion: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013)