Mouse Hepatic Cells Research Articles

Occludin induces microvillus formation via phosphorylation of ezrin in a mouse hepatic cell line

Apical and basolateral cell membranes are separated by tight junctions (TJs). Microvilli are limited to the apical cell membrane. TJs and microvilli are the landmarks for epithelial cell polarity. However, the direct relationship between TJ proteins (TJPs) and the components of microvilli remains unclear. In this study, we investigated whether occludin, which is considered to be a functional TJP, is involved in microvillus formation. In occludin knockout mouse hepatic cells (OcKO cells), the microvillus density was less than that in wild-type (WT) cells and the length of microvilli was short. Immunoreactivity of ezrin was decreased in OcKO cells compared with that in WT cells. Although there was no change in the expression level of ezrin, phosphorylation of ezrin was decreased in OcKO cells. The microvillus density and the length of microvilli were increased in OcKO cells by transfection of full-length mouse occludin and COOH-terminal domains of occludin. These results suggested that occludin induced microvillus formation via phosphorylation of ezrin and that the COOH-terminal domain of occludin, which is localized in non-TJ areas, might be able to induce microvilli formation. Our results provide new insights into the function of occludin.

The Foxo1-Inducible Transcriptional Repressor Zfp125 Causes Hepatic Steatosis and Hypercholesterolemia

Liver-specific disruption of the type 2 deiodinase gene (Alb-D2KO) results in resistance to both diet-induced obesity and liver steatosis in mice. Here, we report that this is explained by an ∼60% reduction in liver zinc-finger protein-125 (Zfp125) expression. Zfp125 is a Foxo1-inducible transcriptional repressor that causes lipid accumulation in the AML12 mouse hepatic cell line and liver steatosis in mice by reducing liver secretion of triglycerides and hepatocyte efflux of cholesterol. Zfp125 acts by repressing 18 genes involved in lipoprotein structure, lipid binding, and transport. The ApoE promoter contains a functional Zfp125-binding element that is also present in 17 other lipid-related genes repressed by Zfp125. While liver-specific knockdown of Zfp125 causes an "Alb-D2KO-like" metabolic phenotype, liver-specific normalization of Zfp125 expression in Alb-D2KO mice rescues the phenotype, restoring normal susceptibility to diet-induced obesity, liver steatosis, and hypercholesterolemia.

Long non-coding RNA cox-2 prevents immune evasion and metastasis of hepatocellular carcinoma by altering M1/M2 macrophage polarization.

Macrophages have been shown to demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. We investigate long non-coding RNA (lncRNA) cox-2 in macrophage polarization and the regulatory mechanism functions in hepatocellular carcinoma (HCC). Lipopolysaccharide (LPS) was used to induce RAW264.7 macrophages into M1 type, and IL-4 was to induce RAW264.7 macrophages into M2 type. We selected mouse hepatic cell line Hepal-6 and hepatoma cell line HepG2 for co-incubation with M1 or M2 macrophages. Quantitative real-time PCR was used to detect the expressions of lncRNA cox-2 and mRNAs. ELISA was conducted for testing IL-12 and IL-10 expressions; Western blotting for epithelial mesenchymal transition related factors (E-cadherin and Vimentin). An MTT, colony formation assay, flow cytometry, transwell assay, and stretch test were conducted to test cell abilities. The M1 macrophages had higher lncRNA cox-2 expression than that in the non-polarized macrophages and M2 macrophages. The lncRNA cox-2 siRNA decreased the expression levels of IL-12, iNOS, and TNF-α in M1 macrophages, increased the expression levels of IL-10, Arg-1, and Fizz-1 in M2 macrophages (all P < 0.05). The lncRNA cox-2 siRNA reduces the ability of M1 macrophages to inhibit HCC cell proliferation, invasion, migration, EMT, angiogenesis and facilitate apoptosis while strengthening the ability of M2 macrophages to promote proliferation HCC cell growth and inhibit apoptosis. These findings indicate that lncRNA cox-2 inhibits HCC immune evasion and tumor growth by inhibiting the polarization of M2 macrophages.

Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12

To develop an all-in-one CRISPR/Cas9 vector system that can efficiently knockdown miR-101a expression in mice. Three sgRNAs targeting mouse miR-101a gene and a small guide (sgRNA) targeting green fluorescent protein gene were designed and constructed into an all-in-one vector system (pENTRY-U6-sgRNA-WT Cas9). Moreover, sgRNA1 and sgRNA3 were selected and constructed into a double-nicking Cas9 vector (pENTRY-U6-sgRNA-U6-sgRNA-Cas9 D10A). The constructed plasmids were transfected into mouse liver AML12 cells for validation by T7 EndoⅠ(T7EⅠ) 72 h after transfection. The pAD vectors were cloned via the Gateway system, and the recombinant adenovirus vectors were packaged in 293A cells. The virus particles were used to infect AML12 cells and the expression levels of mature miR-101a were analyzed to monitor the knockout efficiency after 72 h. The constructed pENTRY all-in-one vectors were validated by gene sequencing and T7EⅠ assay, which showed CRISPR/Cas9-mediated mismatches at target sites of miR-101a gene. The adenovirus vectors were constructed successfully. The CRISPR/Cas9 containing adenovirus was introduced to AML12 cells and the quantitative real-time PCR assays indicated that the expression level of mature miR-101a was significantly decreased compared with that of the control (all P<0.01). We have successfully constructed two "all-in-one" CRISPR/Cas9 vector systems targeting miR-101a gene in mouse liver AML12 cells with high efficiency. It provides experimental basis for research of microRNA, and a reference method for knockout of other miRNAs.

MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Cyanidin-3-O-β-glucoside combined with its metabolite protocatechuic acid attenuated the activation of mice hepatic stellate cells.

Previous studies indicated that cyanidin-3-O-β-glucoside (C3G) as a classical anthocyanin exerted an anti-fibrotic effect in the liver, but its bioavailability was quite low. This study was undertaken to explore the restraining effect of C3G and its metabolite protocatechuic acid (PCA) on the activation of hepatic stellate cells (HSCs). Our data demonstrated that the treatment of a carbon tetrachloride-treated mice model with C3G inhibited liver fibrosis and HSC activation. In vitro, both C3G and PCA preserved the lipid droplets and retinol in primary HSCs, and additionally inhibited the mRNA expression of α-smooth muscle actin and collagen I, but elevated the level of matrix metalloproteinase-2 and liver X receptors. Only PCA suppressed the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secreted from HSCs significantly. In addition, C3G and PCA inhibited the proliferation and migration of HSCs. In conclusion, PCA mainly explained the in vivo inhibiting effect of C3G on HSC activation and liver fibrosis.

Lipopolysaccharide promotes tumorigenicity of hepatic progenitor cells by promoting proliferation and blocking normal differentiation

Hepatic progenitor cells (HPCs) are bipotential stem cells that can differentiate into mature hepatocytes or biliary epithelial cells (BECs). They are thought to be involved in repair of liver injury and the incidence of hepatic carcinoma. Their physiology is closely associated with the microenvironment where they reside. Lipopolysaccharide (LPS), an important component of the hepatic pathological microenvironment, is stored in the liver and affects many types of cells in various hepatosis. HPCs may also be influenced by LPS. In this paper, mouse ED13.5 E-cadherin+ foetal liver cells were isolated as mouse hepatic progenitor cells (mHPCs). Proliferation of mHPCs was promoted under LPS conditions both in vivo and in vitro. Moreover, LPS enhanced colony formation ability of mHPCs, and blocked them differentiation into mature hepatocytes and formation of a bile duct-liked structure. More importantly, long-term treatment with LPS promoted tumorigenesis of mHPCs in nude mice. Thus, we conclude that LPS may promote aberrant proliferation of mHPCs and restrict their normal differentiation. Long-term exposure of mHPCs to LPS increased the risk of tumour formation. These data provide insight into the links between LPS, HPCs fate, and tumorigenesis, and present novel insight into the relationship between HPCs and their microenvironment.

MicroRNAs miR-125b and miR-100 suppress metastasis of hepatocellular carcinoma by disrupting the formation of vessels that encapsulate tumour clusters.

We have previously shown that vessels that encapsulated tumour cluster (VETC), a prevalent vascular pattern in hepatocellular carcinoma (HCC), facilitates the entry of the whole tumour cluster into the bloodstream in an invasion-independent manner, and that angiopoietin 2 (Angpt2), the levels of which are increased in HCC cells, is essential for VETC formation. However, the mechanisms underlying VETC formation remains unclear. Herein, we characterized miR-125b and miR-100 as novel VETC suppressors by using human HCC specimens, and cell and animal models. We showed that reduced expression of either miR-125b or miR-100 in human HCC tissues was significantly associated with the presence of VETC, venous invasion of tumour cells, and the occurrence of endothelium-coated microemboli. To confirm the role of miR-125b and miR-100 in VETC formation and HCC metastasis, cell lines with stable miR-125b and miR-100 expression were established by using human VETC-2 cells and mouse Hepa1-6 cells, the hepatoma cells that developed xenografts with VETC patterns. Our results showed that expression of miR-125b or miR-100 in VETC-2 and Hepa1-6 cells dramatically reduced VETC formation in xenografts, and consequently inhibited in vivo metastasis, suggesting that miR-125b and miR-100 may attenuate metastasis by repressing VETC formation. Further investigation revealed that miR-125b directly suppressed the expression of Angpt2 by binding to its 3'-untranslated region, whereas miR-100 reduced the protein level of Angpt2 by targeting mechanistic target of rapamycin (MTOR) and blocking the MTOR-p70S6K signalling pathway. Moreover, the suppressive effect of miR-125b and miR-100 on VETC formation was abrogated by injecting Angpt2-expressing viruses into xenografts. Taken together, our findings imply that miR-125b and miR-100 negatively regulate Angpt2 expression through different mechanisms, in turn inhibit VETC formation, and consequently abrogate the VETC-dependent metastasis of hepatoma cells. This study uncovers new regulatory mechanisms of VETC formation, identifies novel functions of miR-125b and miR-100, and provides new targets for antimetastasis therapy of HCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enhanced transgene expression by plasmid-specific recruitment of histone acetyltransferase

Histone acetylation is associated with the activation of genes on chromosomes. Transgene expression from plasmid DNA might be increased by the acetylation of histones bound to plasmid DNA. To examine this hypothesis, we employed a positive feedback system, using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the histone acetyltransferase (HAT) domain of mouse CREB-binding protein (GAL4-HAT), in which GAL4-HAT promotes its own expression as well as that of a reporter gene product (luciferase). The activator plasmid DNA carrying the gene encoding GAL4-HAT was introduced into mouse Hepa1-6 cells, together with the reporter plasmid DNA, by lipofection. Significantly increased luciferase expression was observed by the co-introduction of the activator plasmid DNA. Moreover, the acetylation of histones bound to the reporter plasmid DNA was enriched by the activator plasmid DNA. These results indicated that the GAL4-HAT system is useful for enhanced transgene expression.

MiR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

RationaleRecently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and resultsOur results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. ConclusionsThe results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.

Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

A Cytochrome P450‐independent Mechanism of Acetaminophen‐induced Hepatocellular Injury

Acetaminophen (APAP) hepatotoxicity in mice resembles in most ways that which occurs in humans. Primary mouse hepatic parenchymal cells (HPCs) are the most frequently used cell type in vitro to study mechanisms of APAP hepatotoxicity. It is widely accepted that APAP hepatotoxicity requires bioactivation by cytochromes P450 (CYPs), but this remains unproven in HPCs over the wide range of concentrations that have been used in published reports. Accordingly, we tested the hypothesis that APAP‐induced HPC death in vitro depends solely on CYPs. We evaluated APAP cytotoxicity and bioactivation (formation of APAP‐protein adducts) in the presence and absence of the CYP inhibitor 1‐aminobenzotriazole (1‐ABT). 1‐ABT abolished formation of APAP‐protein adducts at all concentrations tested (0.1‐14mM) but only eliminated cytotoxicity at small APAP concentrations (0.1‐5mM), indicating the presence of a CYP‐independent mechanism at larger concentrations. The APAP deacetylation product, p‐aminophenol (PAP), was detected in HPCs exposed to a large APAP concentration, and a deacetylase inhibitor significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a CYP‐dependent mechanism that operates at smaller APAP concentrations and a CYP‐independent mechanism that predominates at larger concentrations and is later in onset. PAP likely contributes to the latter mechanism. (Supported by NIH grant R01 DK087886)

Shotgun metabolomic approach based on mass spectrometry for hepatic mitochondria of mice under arsenic exposure.

Mass spectrometry (MS)-based toxicometabolomics requires analytical approaches for obtaining unbiased metabolic profiles. The present work explores the general application of direct infusion MS using a high mass resolution analyzer (a hybrid systems triple quadrupole-time-of-flight) and a complementary gas chromatography-MS analysis to mitochondria extracts from mouse hepatic cells, emphasizing on mitochondria isolation from hepatic cells with a commercial kit, sample treatment after cell lysis, comprehensive metabolomic analysis and pattern recognition from metabolic profiles. Finally, the metabolomic platform was successfully checked on a case-study based on the exposure experiment of mice Mus musculus to inorganic arsenic during 12 days. Endogenous metabolites alterations were recognized by partial least squares-discriminant analysis. Subsequently, metabolites were identified by combining MS/MS analysis and metabolomics databases. This work reports for the first time the effects of As-exposure on hepatic mitochondria metabolic pathways based on MS, and reveals disturbances in Krebs cycle, β-oxidation pathway, amino acids degradation and perturbations in creatine levels. This non-target analysis provides extensive metabolic information from mitochondrial organelle, which could be applied to toxicology, pharmacology and clinical studies.

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20min. Precision in terms of relative standard deviation (n=5) is of 3–5% and detection limits is 0.21ngCug−1.The application of the methodology to hepatic cells from mice exposed to inorganic mercury reveals decreased levels of Cu,Zn-SOD in cytosolic and mitochondrial extracts, as a consequence of the oxidative stress caused by this toxic metal. Additionally, the quantification of mitochondrial Cu,Zn-SOD in hepatic cells from Mus musculus has been carried out for the first time.

Development of Hepatocellular Carcinoma in a Murine Model of Nonalcoholic Steatohepatitis Induced by Use of a High-Fat/Fructose Diet and Sedentary Lifestyle

Obesity is increasingly prevalent, strongly associated with nonalcoholic liver disease, and a risk factor for numerous cancers. Here, we describe the liver-related consequences of long-term diet-induced obesity. Mice were exposed to an extended obesity model comprising a diet high in trans-fats and fructose corn syrup concurrent with a sedentary lifestyle. Livers were assessed histologically using the nonalcoholic fatty liver disease (NAFLD) activity score (Kleiner system). Mice in the American Lifestyle-Induced Obesity Syndrome (ALIOS) model developed features of early nonalcoholic steatohepatitis at 6 months (mean NAFLD activity score = 2.4) and features of more advanced nonalcoholic steatohepatitis at 12 months, including liver inflammation and bridging fibrosis (mean NAFLD activity score = 5.0). Hepatic expression of lipid metabolism and insulin signaling genes were increased in ALIOS mice compared with normal chow-fed mice. Progressive activation of the mouse hepatic stem cell niche in response to ALIOS correlated with steatosis, fibrosis, and inflammation. Hepatocellular neoplasms were observed in 6 of 10 ALIOS mice after 12 months. Tumors displayed cytological atypia, absence of biliary epithelia, loss of reticulin, alteration of normal perivenular glutamine synthetase staining (absent or diffuse), and variable α-fetoprotein expression. Notably, perivascular tumor cells expressed hepatic stem cell markers. These studies indicate an adipogenic lifestyle alone is sufficient for the development of nonalcoholic steatohepatitis, hepatic stem cell activation, and hepatocarcinogenesis in wild-type mice.

Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: Role of E2F1 and transcription factor dimerization partner 2

c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3β, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3β expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

Wnt3-induced-epithelial-mesenchymal transition in mouse hepatic progenitor cells

Objective To investigate the impact of wnt3upon epithelial- mesenchymal transition in mouse hepatic progenitor cells(14-19cells).Methods Ad-GFP-wnt3virus and blank Ad-GFP were transfected into 14-19cells;cell morphology was observed under microscope.The migration ability of 14-19cells was examined by wound-healing assay and transwell assay.The expression of epithelial and mesenchymal markers in 14-19cells was detected by real-time PCR and Western blotting analysis.Results Microscopic observation found that 14-19cells changed from an epithelial characteristic shape into a spindle-like shape.Wound-healing assay and Transwell assay showed that the migration ability of 14-19cells highly expressing wnt3was significantly enhanced compared with the blank control group(P0.01).Real-time PCR and Western blotting analysis showed that mesenchymal markers N-cadherin,vimentin and twist1were significantly increased at both mRNA and protein levels in Ad-GFP-wnt3virus group than the blank control group,while the epithelial marker Ecadherin was significantly decreased at mRNA and protein levels(P0.01).Conclusion Wnt3can induce epithelialmesenchymal transition in mouse hepatic progenitor cells,indicating that wnt3may take part in the progress of hepatic fibrosis.

Oxidative-damage effect of Fe3O4 nanoparticles on mouse hepatic and brain cells in vivo

To assess the biological safety of Fe3O4 nanoparticles (NPs), the oxidative-damage effect of these NPs was studied. Twenty-five Kunming mice were exposed to Fe3O4 NPs by intraperitoneal injection daily for 1 week at doses of 0, 10, 20, and 40 mg·kg−1. Five Kunming mice were also injected with 40 mg·kg−1 ordinary Fe3O4 particles under the same physiological conditions. Biomarkers of reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA) in the hepatic and brain tissues were detected. Results showed that no significant difference in oxidative damage existed at concentrations lower than 10 mg·kg−1 for NPs compared with the control group. Fe3O4 NP concentration had obvious dose-effect relationships (P < 0.05 or P < 0.01) with ROS level, GSH content, and MDA content in mouse hepatic and brain tissues at > 20mg·kg−1 concentrations. To some extent, ordinary Fe3O4 particles with 40 mg·kg−1 concentration also affected hepatic and brain tissues in mice. The biological effect was similar to Fe3O4 NPs at 10 mg·kg−1 concentration. Thus, Fe3O4 NPs had significant damage effects on the antioxidant defense system in the hepatic and brain tissues of mice, whereas ordinary Fe3O4 had less influence than Fe3O4 NPs at the same concentration.

Control of Liver Tissue Reconstitution in Mesenteric Leaves: The Effect of Preculture on Mouse Hepatic Progenitor Cells Prior to Transplantation

Our objective is to control the reconstitution of liverlike tissues at extrahepatic sites using hepatic progenitor cells (HPCs) andin vitropreculture prior to transplantation. We prepared cell-based hybrid grafts by culturing HPCs isolated from fetal E14.5 mouse livers on biodegradable, highly porous 3-dimensional poly-L-lactic acid (PLLA) scaffolds for 1 week in basal medium (the basal condition) or 10 mM nicotinamide (NA) and 1% dimethyl sulfoxide (DMSO) supplemented conditions (the ND-positive condition) prior to implantation. Sections of hybrid grafts cultured for 1 week showed that HPCs grew and spread on the surface of scaffolds under both basal and ND (+) conditions. Most of these cells were albumin (+) and CK18 (+). CK19 (+) cells were also present under the basal condition but not the ND (+) condition. Cultured hybrid grafts were implanted into the mesenteric leaves of mice and removed after 1 month. Transplanted tissues cultured under the basal condition consisted of albumin (+) hepatocyte-like and CK19 (+) biliary epithelial cell (BEC)-like cells organized in duct-like structures. In contrast, integrated tissues cultured under the ND (+) condition alone had differentiated albumin (+) hepatocyte-like cells and were relatively larger than those under the basal condition. Hepatocyte-like cells of transplanted hybrid grafts cultured under both conditions were periodic acid-Schiff (PAS) staining-positive and expressed transcription factors, hepatocyte nuclear factor (HNF) 4 and CCAAT/enhancer-binding protein (C/EBP) α. These findings suggest that combining progenitor cells andin vitropreculture may potentially regulate liverlike tissues at extrahepatic sites.