Hepa1-6 Cells Research Articles

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40-50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

Targeted transplantation of mitochondria to hepatocytes.

BackgroundMitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis.Materials and methodsAn AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release.ResultsCoincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days.ConclusionRescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis.

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.

Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47 phox phosphorylation of NADPH oxidase in SK Hep-1 cells.

Baccharis trimera, popularly known as "carqueja", is a native South-American plant possessing a high concentration of polyphenolic compounds and therefore high antioxidant potential. Despite the antioxidant potential described for B. trimera, there are no reports concerning the signaling pathways involved in this process. So, the aim of the present study was to assess the influence of B. trimera on the modulation of PKC signaling pathway and to characterize the effect of the nicotinamide adenine dinucleotide phosphate oxidase enzyme (NOX) on the generation of reactive oxygen species in SK Hep-1 cells. SK-Hep 1 cells were treated with B. trimera, quercetin, or rutin and then stimulated or not with PMA/ionomycin and labeled with carboxy H2DCFDA for detection of reactive oxygen species by flow cytometer. The PKC expression by Western blot and enzyme activity was performed to evaluate the influence of B. trimera and quercetin on PKC signaling pathway. p47 phox and p47 phox phosphorylated expression was performed by Western blot to evaluate the influence of B. trimera on p47 phox phosphorylation. The results showed that cells stimulated with PMA/ionomycin (activators of PKC) showed significantly increased reactive oxygen species production, and this production returned to baseline levels after treatment with DPI (NOX inhibitor). Both B. trimera and quercetin modulated reactive oxygen species production through the inhibition of PKC protein expression and enzymatic activity, also with inhibition of p47 phox phosphorylation. Taken together, these results suggest that B. trimera has a potential mechanism for inhibiting reactive oxygen species production through the PKC signaling pathway and inhibition subunit p47 phox phosphorylation of nicotinamide adenine dinucleotide phosphate oxidase.

P36 Pericyte seeded biofunctionalised vascular graft for improved endothelial coverage

Coronary bypass is the most common intervention for coronary obstruction. When autologous vessels are unavailable, synthetic grafts are employed. The challenge of rapid endothelialisation to reduce complications is still far from being achieved. This study describes a novel approach to synthetic graft design, based on the spatially defined presentation of functionalities within the scaffold, contributing to the biological activity of the pre-seeded pro-angiogenic human saphenous vein-derived pericytes (SVP). Indeed, the electrospun polycaprolactone (PCL) presented two distinct functionalities on either side: the osteopontin-derived adhesion peptide (Adh) to facilitate endothelial adhesion on the luminal side and the heparin binding peptide (Hep) to create a reservoir of SVP-produced growth factors on the adventitial side. Conjugated peptides retained functionality within the scaffold, with Adh increasing endothelial cells (EC) adhesion and Hep binding the SVP-produced VEGF and releasing it to improve EC growth over time. Additionally, seeding of the scaffold with SVP increased EC migration and proliferation, suggesting their supporting role in re-endothelialisation of the graft. When seeded on the dual peptide scaffold, SVP acted synergistically with the peptides determining a greater EC coverage, as compared to each component alone. SVP-seeded scaffolds were cryopreserved for up to 2 weeks in GMP-grade medium preserving cell viability and functionality. In conclusion, we engineered a progenitor cell-seeded vascular graft, which incorporates spatially organised biomolecules that act synergistically with the cells to support EC coverage.

Adenovirus-mediated interleukin 21 gene transfer enhances antitumor immunity and reduces tumorigenicity of Hepa1-6 in mice.

In the present study, adenovirus-mediated interleukin 21 (Ad5-IL-21-EGFP) gene expression was induced in Hepa1–6 cells to investigate whether IL-21 was capable of enhancing antitumor immunity and reducing tumorigenicity of Hepa1–6 in a mouse model. Mice were inoculated intradermally into the right flank with Hepa1–6 cells or Hepa1–6 cells infected with Ad5 or Ad5-IL-21. Four weeks later, the mice were sacrificed humanely, and the tumor volume, tumor weight and mouse spleen index were measured. The levels of IL-21, IL-4 and interferon (IFN)-γ levels in mouse serum and tumor tissues were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Cell counting kit-8 (CCK-8) assay was used to detect the killing ability of spleen T cells and natural killer (NK) cells, and the proliferation ability of T cells. The expression of IL-21 was confirmed by reverse transcription-polymerase chain reaction, western blot analysis and ELISA assay in Ad5-IL-21-EGFP-infected Hepa1–6 cells. The overexpression of IL-21 significantly reduced the tumorigenicity of Hepa1–6 cells. The tumor volumes and tumor weights in Ad5-IL-21-Hepa1–6 mice were much smaller than those in the Ad5-Hepa1–6 group and Hepa1–6 wild-type group. The immunohistochemistry and ELISA assay demonstrated that IL-21 and IFN-γ levels were much higher while the IL-4 level was much lower in the Ad5-IL-21-Hepa1–6 group than in the other two groups. CCK-8 assay revealed that the killing ability of NK cells and T cells, and the proliferation ability of T cells in Ad5-IL-21-Hepa1–6 mice were higher than in the other two groups; the spleen index of Ad5-IL-21-Hepa1–6 mice was also higher than in the other groups. The data had a significant difference (P<0.01). In conclusion, IL-21 reduces tumorigenicity of Hepa1–6 by a mechanism involving enhanced activation of cell-mediated immunity in tumor-bearing mice.

Vitexin suppresses autophagy to induce apoptosis in hepatocellular carcinoma via activation of the JNK signaling pathway.

Vitexin, a flavonoids compound, is known to exhibit broad anti-oxidative, anti-inflammatory, analgesic, and antitumor activity in many cancer xenograft models and cell lines. The purpose of this study was to investigate the antitumor effects and underlying mechanisms of vitexin on hepatocellular carcinoma. In this study, we found that vitexin suppressed the viability of HCC cell lines (SK-Hep1 and Hepa1-6 cells) significantly. Vitexin showed cytotoxic effects against HCC cell lines in vitro by inducing apoptosis and inhibiting autophagy. Vitexin induced apoptosis in a concentration-dependent manner, and caused up-regulations of Caspase-3, Cleave Caspase-3, and a down-regulation of Bcl-2. The expression of autophagy-related protein LC3 II was significantly decreased after vitexin treatment. Moreover, western blot analysis presented that vitexin markedly up-regulated the levels of p-JNK and down-regulated the levels of p-Erk1/2 in SK-Hep1 cells and Hepa1-6 cells. Cotreatment with JNK inhibitor SP600125, we demonstrated that apoptosis induced by vitexin was suppressed, while the inhibition of autophagy by vitexin was reversed. The results of colony formation assay and mouse model confirmed the growth inhibition role of vitexin on HCC in vitro and in vivo. In conclusion, vitexin inhibits HCC growth by way of apoptosis induction and autophagy suppression, both of which are through JNK MAPK pathway. Therefore, vitexin could be regarded as a potent therapeutic agent for the treatment of HCC.

Combined incubation of cadmium, docosahexaenoic and eicosapentaenoic acid affecting the oxidative stress and antioxidant response in human hepatocytes in vitro.

Human hepatocellular cells Hep G2 were used to investigate the effects of the intake of contaminated fish on oxidative stress. Uptake of heavy metal contaminated fish was mimicked by incubating the cells with a combination of cadmium chloride (Cd(2+)) as possible contaminant and a combination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as important fatty acids (FA) specific for fish. The main aim of this study was to determine the effects of these co-incubations (FA, Cd(2+)) on lipid and protein oxidation. In addition we also evaluated the antioxidant response of the cells using two different methods (SOD and TAC). Pre-incubation with the chosen FA significantly reduced the oxidative stress caused by incubation with Cd(2+). We measured an increased level of carbonyl proteins (CP) in the cells pre-incubated with bovine serum albumin (BSA) and post-incubated with Cd(2+).

Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism.

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns.

Abstract 4068: Role of cytochrome P450 (CYP)1A2 in the molecular regulation of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1)

Abstract Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC withdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in Cyp1a2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 induction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this phenomenon. Hepa-1 cells were treated with the MC (2.5 μM), or dimethylsulfoxide (DMSO) as control, and at selected time points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. A 24 h treatment induced the CYP1A1 promoter activity, CYP1A1 mRNA, and ethoxyresorufin O-deethylase (EROD) activity by 2-, 1000-, and 15-fold, respectively. The induction was sustained for 4-5 days. MC persistently induced CYP1A1 apoprotein level as well. Electrophoretic mobility shift assay indicated that MC induced a nuclear protein that bound to aryl hydrocarbon response elements in the CYP1A1 promoter region. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter activity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly attenuated by CYP1A2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. Citation Format: Sudha Kondraganti, Chun Chu, Bhagavatula Moorthy. Role of cytochrome P450 (CYP)1A2 in the molecular regulation of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4068.

Abstract LB-005: TRIP13/PCH2 is a novel regulator of EGF-mTOR signaling promoting hepatocellular carcinoma development

Abstract Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related mortality with limited treatment options and bleak prognosis. The main risk factors for HCC have been linked to hepatitis infection, alcoholic liver injury, non-alcoholic fatty liver disease (NAFLD) and metabolic diseases. Current therapeutic options are limited, which could be due to the lack of understanding of the key cellular players and signalling pathways in HCC. In order to find novel regulators of HCC development, we surveyed expression data of human hepatocellular carcinoma (E-GEOD-25097) and found the AAA-ATPase Thyroid Hormone Receptor Interactor 13 (TRIP13) to be upregulated in tumorous tissue compared to non-tumorous tissue. Consistently, using a mouse model for HCC by injection of Diethylnitrosamine (DEN), we observed induced expression of TRIP13 in liver tumors compared to healthy liver tissue. Additionally, in a model of liver regeneration by partial hepatectomy, induction of TRIP13 coincided with a peak in cell proliferation in the regenerating liver. Strinkingly, Adeno-associated virus (AAV)-mediated hepatocyte-specific knockdown of TRIP13 impeded liver regeneration upon partial hepatectomy, suggesting an important role of TRIP13 in liver cell proliferation. Indeed, knockdown (KD) of TRIP13 severely impaired cell growth and proliferation in different human and murine HCC cell lines in vitro. To investigate the oncogenic potential of TRIP13 in vivo, we injected lentiviral infected cells subcutaneously into mice and measured tumor growth. Remarkably, knockdown of TRIP13 in Hepa1-6 cells led to the development of smaller tumors compared to control cells, whereas TRIP13-deficient Huh7 cells did not form any tumors. Interestingly, shRNA knockdown of TRIP13 in HCC cell lines showed decreased phosphorylation of key players in the mTOR signalling pathway and reduction of the downstream Cyclin D1 levels. Concurrently, the mRNA and protein expression of EGF receptor was also downregulated in TRIP13 KD-cells. Taken together, we identified a novel role of TRIP13 during hepatocyte proliferation both in liver regeneration and HCC. Additionally, our findings also support an oncogenic function of TRIP13 through the regulation of the EGFR-mTOR signalling axis. Citation Format: Bettina Meissburger, Jessica Chan, Roldan de Guia, Stephan Herzig, Mauricio Berriel Diaz. TRIP13/PCH2 is a novel regulator of EGF-mTOR signaling promoting hepatocellular carcinoma development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-005.

Regulatory effect of microRNA-141 on synthesis of high mobility group protein B1 in mouse carcinoma cell line hepal-6

To explore the regulatory effect of microRNA-141 (miR-141) on expression of high mobility group protein B1 (HMGB1) in mouse hepatoma carcinoma cell line (hepal-6 cells). The hepal-6 cells were divided into 4 groups: a miR-141 mimic group, a miR-141 mimic control group, a miR-141 inhibitor group and a miR-141 inhibitor control group. The miR-141 was transfected into hepal-6 cells with lipofectamine 2000. The levels of miR-141 and HMGB1 mRNA in the hepal-6 cells were detected by quantitative real-time PCR (qRT-PCR), and then HMGB1 protein was examined by Western blot. The regulatory effect of miR-141 on 3'-UTR of candidate target gene (HMGB1) was determined by dual-luciferase reporter assay. Compared with the control group, the level of miR-141 was up-regulated in the miR-141 mimic group, while down-regulated in the miR-141 inhibitor group. Moreover, the levels of HMGB1 mRNA and protein decreased in the miR-141 mimic group, while increased in the miR-141 inhibitor group. The dual-luciferase reporter assay showed that miR-141 could target the 3'-UTR of HMGB1 gene. MiR-141 can up-regulate the expression of HMGB1 protein at the post-transcriptional level by targeting to the specific sequence of 3'-UTR of HMGB1 gene, which suggests that HMGB1 gene may be a target gene of miR-141.

Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression

The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling.

Detection of differentially expressed candidate genes for a fatty liver QTL on mouse chromosome 12.

BackgroundThe SMXA-5 mouse is an animal model of high-fat diet-induced fatty liver. The major QTL for fatty liver, Fl1sa on chromosome 12, was identified in a SM/J × SMXA-5 intercross. The SMXA-5 genome consists of the SM/J and A/J genomes, and the A/J allele of Fl1sa is a fatty liver-susceptibility allele. The existence of the responsible genes for fatty liver within Fl1sa was confirmed in A/J-12SM consomic mice. The aim of this study was to identify candidate genes for Fl1sa, and to investigate whether the identified genes affect the lipid metabolism.ResultsA/J-12SM mice showed a significantly lower liver triglyceride content compared to A/J mice when fed the high-fat diet for 7 weeks. We detected differences in the accumulation of liver lipids in response to the high-fat diet between A/J and A/J-12SM consomic mice. To identify candidate genes for Fl1sa, we performed DNA microarray analysis using the livers of A/J-12SM and A/J mice fed the high-fat diet. The mRNA levels of three genes (Iah1, Rrm2, Prkd1) in the chromosomal region of Fl1sa were significantly different between the strains. Iah1 mRNA levels in the liver, kidney, and lung were significantly higher in A/J-12SM mice than in A/J mice. The hepatic Iah1 mRNA level in A/J-12SM mice was 3.2-fold higher than that in A/J mice. To examine the effect of Iah1 on hepatic lipid metabolism, we constructed a stable cell line expressing the mouse Iah1 protein in mouse hepatoma Hepa1-6 cells. Overexpression of Iah1 in Hepa1-6 cells suppressed the mRNA levels of Cd36 and Dgat2, which play important roles in triglyceride synthesis and lipid metabolism.ConclusionsThese results demonstrated that Fl1sa on the proximal region of chromosome 12 affected fatty liver in mice on a high-fat diet. Iah1 (isoamyl acetate-hydrolyzing esterase 1 homolog) was identified as one of the candidate genes for Fl1sa. This study revealed that the mouse Iah1 gene regulated the expression of genes related to lipid metabolism in the liver.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0385-2) contains supplementary material, which is available to authorized users.

Silencing of ST6GalNAc I suppresses the proliferation, migration and invasion of hepatocarcinoma cells through PI3K/AKT/NF-κB pathway.

ST6GalNAc I is the major Sialyl-Tn antigen (STn) synthase that is highly correlated with tumor invasion and metastasis. However, the roles and molecular mechanisms by which ST6GalNAc I mediates the malignant phenotypes of hepatocarcinoma cells still remain poorly unknown. In this study, we investigated the expression of STn and ST6GalNAc I in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have different metastatic potential, as compared with normal mouse liver cell line IAR-20. The results showed that the expression of ST6GalNAc I and STn in Hca-F and Hca-P cells was much higher than that in Hepa1-6 and IAR20 cells. Knockdown of ST6GalNAc I by shRNA in Hca-F cells significantly decreased the expression of STn and inhibited the growth of tumor cells in vitro and in vivo. This reduction of ST6GalNAc I expression also led to the decreased migration and invasion of Hca-F cells. Furthermore, we found that ST6GalNAc I knockdown inhibited the expression levels of PI3k, p-Akt473, p-Akt308, NF-κB, and their downstream molecules. Together, our results suggest a role of ST6GalNAc I in promoting the growth and invasion of hepatocarcinoma cells through regulating PI3K/AKT signaling, and ST6GalNAc I might be a promising marker for the prognosis and therapy of hepatocarcinoma.

Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes.

Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions.

Regulation of autophagy by systemic admission of microRNA-141 to target HMGB1 in l-arginine-induced acute pancreatitis invivo.

MicroRNAs are endogenous, non-coding RNAs of approximately 20-22 nucleotides that regulate gene expression by binding to the 3' untranslated region (UTR) of target mRNAs and can be applied in gene therapy. Acute pancreatitis is an inflammatory pancreatic disease that carries considerable morbidity and mortality. The purpose of this study was to explore the therapeutic potential of microRNA-141 (miR-141) for acute pancreatitis invivo. AP was induced by two hourly intra-peritoneal (i.p.) injections of l-arginine (200mg×2/100g.BW). Control mice received normal saline injections. In a separate group, normal saline, empty adenoviral vector and miR-141 adenoviral vector were given to the mice via tail vein hydrodynamically at 72h before the first l-arginine injection. All the mice were euthanized at 24h after the last l-arginine injection, and the pancreatic tissues were assessed by qRT-PCR, Western blotting and electron microscopy. miR-141 directly inhibited HMGB1 expression in mouse hepal-6 cell. Furthermore, systemic administration of the miR-141 knock-downthe expression of HMGB1 protein and further antagonized the downstream protein Beclin-1, leading to the reduction of autophagosomes and autolysosomes, blockade of the LC3-II level and the increased levels of p62 protein after injection of l-arginine. In addition, the level of Lamp-2 was not significantly different. For the first time miR-141 was applied in acute pancreatitis treatment invivo. Inhibition of HMGB1 by miR-141 may block the process of autophagosome formation through the HMGB1/Beclin-1 pathway. The miR-141 appears to be a promising candidate for the gene therapy of acute pancreatitis.

Selective portal vein injection for the design of syngeneic models of liver malignancy

Liver metastases are the most frequent cause of death due to colorectal cancer (CRC). Syngeneic orthotopic animal models, based on the grafting of cancer cells or tissue in host liver, are efficient systems for studying liver tumors and their (patho)physiological environment. Here we describe selective portal vein injection as a novel tool to generate syngeneic orthotopic models of liver tumors that avoid most of the weaknesses of existing syngeneic models. By combining portal vein injection of cancer cells with the selective clamping of distal liver lobes, tumor growth is limited to specific lobes. When applied on MC-38 CRC cells and their mouse host C57BL6, selective portal vein injection leads with 100% penetrance to MRI-detectable tumors within 1 wk, followed by a steady growth until the time of death (survival ∼7 wk) in the absence of extrahepatic disease. Similar results were obtained using CT-26 cells and their syngeneic Balb/c hosts. As a proof of principle, lobe-restricted liver tumors were also generated using Hepa1-6 (C57BL6-syngeneic) and TIB-75 (Balb/c-syngeneic) hepatocellular cancer cells, demonstrating the general applicability of selective portal vein injection for the induction of malignant liver tumors. Selective portal vein injection is technically straightforward, enables liver invasion via anatomical routes, preserves liver function, and provides unaffected liver tissue. The tumor models are reproducible and highly penetrant, with survival mainly dependent on the growth of lobe-restricted liver malignancy. These models enable biological studies and preclinical testing within short periods of time.