Hepatoma Hepa-1 Cells Research Articles

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3’ end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

P13-024-23 Effects of Myocytes-Derived Exosomes on the Growth of Hepa1-6 Hepatoma Cells

P13-024-23 Effects of Myocytes-Derived Exosomes on the Growth of Hepa1-6 Hepatoma Cells

Multitargeting Strategy Using Tetrathiomolybdate and Lenvatinib: Maximizing Antiangiogenesis Activity in a Preclinical Liver Cancer Model.

The study aims to investigate the suppressing tumor-promoting effects via multi-anti-angiogenesis activity of the copper chelator (ammonium tetrathiomolybdate, TM) combined with lenvatinib for hepatocellular carcinoma. A total of 55 C57 mice were injected subcutaneously with Hepa1-6 hepatoma cell suspensions into the right posterior thigh. After 7 days, the subcutaneous tumors were formed, and the mice were randomly divided into five groups: TM (G1), Lenvatinib (G2), TM+Lenvatinib (G3), Control (G4), and Copper (II) Gluconate (G5). The copper concentrations in serum and tumors were measured at the predetermined time points. After 14 days of treatments, tumor weight and volumes were analyzed, histology was observed, and the expressions of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in tumor tissues were measured by immunohistochemistry. The median concentration of copper in serum was 401.70, 469.40, and 665.35 μg/L in normal mice, in mice 7 days after implantation, and in the control group, respectively. The intratumoral copper concentrations were higher in G4 mice than in mice 7 days after implantation (P < 0.05). The serum concentration of copper was higher in G5 than all the other groups (P < 0.05; (G1, G2, and G3) vs. G4, P < 0.05; G1 vs. G2, P = 0.013; G2 vs. G3, P = 0.018; G1 vs. G3, P = 0.903. The intratumoral copper concentrations were 608.40, 980.00, 539.31, and 2938.90 μg/L in G1, G2, G3, and G5, respectively. The average tumor weight was 0.55, 0.44, 0.08, 1.37, and 3.11 in G1, G2, G3, G4, and G5, respectively. G5 vs. other groups, P < 0.05; (G1, G2, and G3) vs. G4, P < 0.05; G1 vs. G3, P < 0.05; G2 vs. G3, P < 0.05; G1 vs. G2, P > 0.05. Furthermore, the expression levels of VEGF were significantly lower in G1, G2, and G3 than in G4 and G5 (P < 0.05). A similar trend was observed for MVD in the five groups, but no significant difference was detected in G1 and G2. The study showed a significant positive correlation between tumor load and copper. Copper promotes tumor progression, but copper chelating suppresses tumor growth. The combination of TM with lenvatinib reduces tumor angiogenesis and improves the effect of antitumor treatment. These findings underlie the clinical application of combination therapy.

Suppressive Effects of Siegesbeckia orientalis Ethanolic Extract on Proliferation and Migration of Hepatocellular Carcinoma Cells through Promoting Oxidative Stress, Apoptosis and Inflammatory Responses.

Previous studies have demonstrated that Siegesbeckia orientalis (SO) has a suppressive effect on the growth and migration of endometrial and cervical cancer cells. The present study examined the effect of SO ethanolic extract (SOE) on the proliferation and migration of hepatocellular carcinoma (HCC) and examined the effects of SOE on non-cancerous cells using HaCaT keratinocytes as a model. The SOE effectively inhibited the proliferation of Hepa1-6 (IC50 = 282.4 μg/mL) and HepG2 (IC50 = 344.3 μg/mL) hepatoma cells, whereas it has less cytotoxic effect on HaCaT cells (IC50 = 892.4 μg/mL). The SOE treatment increased the generation of ROS in HCC, but decreased the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase. In contrast, it reduced intracellular ROS formation and upregulated the expression of the related antioxidant enzymes in the H2O2-stimulated HaCaT cells. The SOE intervention also down-regulated the anti-apoptotic Bcl-2 and the migration-related proteins including matrix metalloproteinases (MMPs) and β-catenin in the HCC, suggesting that SOE could promote HCC apoptosis and inhibit HCC migration. On the contrary, it reduced apoptosis and promoted the migration of the keratinocytes. Additionally, the SOE treatment significantly up-regulated the pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, in Hepa1-6 and HepG2 cells. Conversely, it significantly decreased the expression of these cytokines in the H2O2-induced HaCaT cells. These findings indicated that SOE treatment can delay the progression of HCC by increasing oxidative stress, promoting inflammatory response, inducing cancer cell apoptosis and inhibiting their migration. It also has protective effects from pro-oxidant H2O2 in non-cancerous cells. Therefore, SOE may provide a potential treatment for liver cancer.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

BackgroundAberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA.MethodsCreation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors.ResultsComparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects.Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA.ConclusionsSubtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Abstract 2894: Intracellular gap formation of the liver sinusoidal endothelial cells facilitates the liver metastasis ability of cancer cells

Abstract Engraftment of cancer cells to liver pre-metastatic niches and their subsequent growth requires complex interaction with liver sinusoidal endothelial cells (LSECs). Intracellular gap formation of LSECs results from the destruction of the fenestration under histopathological conditions, but their role in liver metastasis is not fully understood. Therefore, this study aims to clarify the molecular function of the gap formation in liver metastasis. To study gap formation, acetaminophen (APAP) and thioacetamide (TAA) were used to pretreat C57BL/6 wild-type mice by intraperitoneal injection. Then, 2 x 106 mouse hepatoma Hepa1-6 cells and colon cancer Colon-26 cells were intrasplenically injected in both treated and non-treated groups. Mice were sacrificed after 18 hours and 72 hours of injection to observe the cancer cell invasion in the hepatic sinusoidal lumen and to count the number of cancer cell engraftment in the liver, respectively. Hepa1-6 cells and isolated primary mouse LSECs were co-cultured for 18 hours to observe the phenomenon in vitro. Quantitative-PCR, Hematoxylin &amp; Eosin, immunofluorescence staining, scanning electron microscope (SEM), transmission electron microscope (TEM), and 3 dimensions tomographic reconstruction image (3D-TRI) were conducted to analyze the data. After 72 hours of injection, the number of cancer cells engrafted into the liver was significantly increased in the APAP-injected group compared to the control (p&amp;lt;0.05). In the TAA model, defenestration of LSECs appeared in the fibrotic area whereas gap formation appeared in the non-fibrotic area, cancer cells dominantly engrafted into the non-fibrotic area. In vitro, LSECs co-cultured with Hepa1-6 increased mRNA levels of matrix metalloproteinase-9 (30-fold) and intercellular adhesion molecule 1 (1.5-fold) and depolymerization of F-actin. Simultaneously, the number of fenestrae and gap formation in LSECs was significantly increased compared to monoculture. Both in vivo-, in vitro- SEM and TEM images clearly showed that Hepa1-6 cells induced the gap formation in LSECs and their protrusions extended to the gaps. Furthermore, 3D-TRI reconstructed the image of cancer cells that broke through the LSEC wall and intracellularly invaded the hepatocyte. Our study demonstrated that the formation of intracellular gaps in LSECs promotes the invasion of cancer cells into the liver. This new insight may lead to novel strategies to prevent liver metastasis. Citation Format: Truong Huu Hoang, Misako Sato-Matsubara, Yuasa Hideto, Tsutomu Matsubara, Hayato Urushima, Thuy Thi Thanh Le, Atsuko Daikoku, Yoshinori Okina, Katsutoshi Yoshizato, Norifumi Kawada, Akihiro Tamori. Intracellular gap formation of the liver sinusoidal endothelial cells facilitates the liver metastasis ability of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2894.

NCOA4 Acts in Hepatocytes to Regulate Ferritin Subunit Levels and Hepatic Iron Storage

The intracellular protein nuclear receptor coactivator 4 (NCOA4) has been shown to mediate the degradation of the iron storage protein ferritin (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014). Accordingly, mice with global disruption of Ncoa4 show ferritin accumulation in multiple organs (Bellelli R et al., Cell Rep 2016). How NCOA4 activity in different tissues contributes to local and systemic iron homeostasis, however, has not yet been clearly defined. Here we selectively dissect the role of NCOA4 in the liver, the body's major iron depot.To facilitate targeted Ncoa4 knockdown in hepatocytes, we utilized Ncoa4 -targeting siRNA that was conjugated to triantennary N-acetylgalactosamine (GalNAc- Ncoa4 siRNA) to promote uptake by the hepatocyte asialoglycoprotein receptor. We first validated the ability of GalNAc- Ncoa4 siRNA treatment to target endogenous murine Ncoa4 mRNA for degradation in murine Hepa1-6 hepatoma cells. We next assessed the ability of GalNAc- Ncoa4 siRNA to acutely lower Ncoa4 expression in mouse liver. Eight-week-old C57BL/6N female mice were injected subcutaneously with 1, 3, or 5 mg/kg body weight of a sterile, endotoxin-tested solution of GalNAc- Ncoa4 siRNA in saline or with vehicle (saline) alone, and tissues were collected for analysis 24 hrs after injection. Compared to vehicle alone, GalNAc- Ncoa4 siRNA effectively lowered Ncoa4 expression in total liver RNA; because the degree of Ncoa4 knockdown that was achieved by the 3 mg/kg and 5 mg/kg doses was similar, the 3mg/kg dose was selected for our further experiments to reduce the possibility of off-target effects.We next examined the durability of hepatocyte Ncoa4 knockdown achieved by a single injection of GalNAc -Ncoa4 siRNA as well as the resulting effects on hepatic and systemic iron parameters. Eight-week-old C57BL/6N female mice were injected once with GalNAc- Ncoa4 siRNA or saline vehicle, and blood and tissues were collected for analysis at 1, 2, 3, or 4 weeks after injection. Compared to saline treatment, a single injection of GalNAc -Ncoa4 siRNA produced a significant, sustained reduction in hepatic Ncoa4 mRNA; Ncoa4 mRNA remained maximally suppressed at the 1, 2, and 3-week timepoints and began to rise slightly 4 weeks after injection. Saline-treated mice showed a slight, progressive increase in mean liver non-heme iron concentration (LIC) over the 4 weeks following injection, consistent with the known effect of age on murine hepatic iron stores. However, in mice injected with a single dose of GalNAc- Ncoa4 siRNA, the rise in LIC over the 4-week time period was significantly greater, consistent with increased hepatic iron storage. Immunoblotting of liver lysates revealed that levels of the ferritin heavy (FTH) and light (FTL) subunits increased in mice treated with GalNAc- Ncoa4 siRNA compared to saline-treated controls. These increases in FTH and FTL were evident 1 week after GalNAc- Ncoa4 siRNA injection, and FTH and FTL levels continued to rise with additional weeks of Ncoa4 knockdown. In mice treated with saline, spleen non-heme iron concentration (SIC) increased over the 4 weeks following injection, consistent with the known effect of age on murine splenic iron stores. However, at the 3 and 4-week time points, mice treated with GalNAc- Ncoa4 siRNA showed a significant reduction in SIC compared to saline-treated controls, which correlated histologically with reduced iron retention in splenic macrophages. Compared to saline treatment, GalNAc -Ncoa4 siRNAtreatment did not produce significant changes in spleen weight, serum iron, complete blood count, or non-heme iron concentration of heart, pancreas, or muscle.Collectively, our findings indicate that NCOA4 acts in hepatocytes to limit LIC as well as hepatic levels of FTH and FTL. Furthermore, local NCOA4 activity in hepatocytes impacts iron distribution to the spleen. Our results are compatible with a model in which degradation of the ferritin protein complex in hepatocytes occurs as a constitutive process that is mediated by NCOA4. Importantly, our findings demonstrate that in the setting of hepatocyte Ncoa4 knockdown, other cellular pathways cannot compensate to maintain normal LIC and normal hepatic levels of FTH and FTL. Additionally, our findings raise the possibility that enhancement of NCOA4 activity in hepatocytes might serve as a therapeutic approach to promote hepatic iron mobilization in iron overload disorders. DisclosuresNo relevant conflicts of interest to declare.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

Effect of sidt2 Gene on Cell Insulin Resistance and Its Molecular Mechanism.

Background Sidt2 (SID1 transmembrane family, member 2) is a multiple transmembrane lysosomal membrane protein newly discovered in our previous study. In the previous study, we used gene targeting technique to make a mouse model of sidt2 gene knockout (sidt2−/−). It was found that sidt2−/− mice showed elevated fasting blood glucose and impaired glucose tolerance, showing a disorder of glucose metabolism, suggesting that sidt2 may be closely related to insulin resistance. We used 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells as subjects to observe the effects of sidt2 on insulin-stimulated glucose uptake and the abovementioned insulin signal transduction pathways, and then to explore the effect of sidt2 on peripheral tissue insulin resistance and its possible molecular mechanism. Methods (1) Lentiviruses with sidt2 gene knockout and puromycin resistance were constructed by Crispr/cas9 vector and transfected into 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells to construct sidt2 knockout cell line model. (2) Glucose uptake of 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells stimulated by insulin was detected by glucose detection kit, and the results were analyzed. (3) Sidt2 knockout group and control group of 3T3-L1 adipocytes, C2-C12 myoblast, and HEPA1-6 hepatoma cells were cultured according to the routine method. The total proteins of the above cells were extracted, and the expression of PAKT (thr308), PI3-K, and PIRS-1 (ser307) in the IRS-1 signaling pathway of the three groups was detected by western blot technique. Results (1) The sidt2 elimination models of 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells were successfully constructed. (2) It was found that the glucose uptake of cells in the sidt2 knockout group was lower than that in normal group under insulin stimulation through the detection of glucose concentration in the cell culture medium. (3) It was found that the expression of PAKT (thr308) and PI3-K protein decreased and the expression of PIRS-1 (ser307) protein increased in sidt2−/− group compared to the control group. Conclusions sidt2 knockout can reduce glucose uptake in peripheral tissue under insulin stimulation, which may lead to peripheral tissue insulin resistance by affecting the IRS-1 signal pathway.

Stem cell factor produced by tumor cells expands myeloid-derived suppressor cells in mice

Immunotherapy is a novel treatment approach for cancers; however, its therapeutic effects are impeded by myeloid-derived suppressor cells (MDSCs). This study aimed to determine how MDSCs are expanded in cancer hosts. MDSCs were positive for Gr-1 and CD11b. Hepa1-6 hepatoma cells, EL4 lymphoma cells, and mice bearing Hepa1-6 hepatoma or lymphoma were examined. Following the inoculation of Hepa1-6 cells into the flanks of mice, a linear correlation was evident between the frequency of MDSCs in the spleen and tumor sizes. MDSC numbers diminished gradually and returned to the normal level within 3 weeks if the tumors were excised. To identify the cytokines produced by tumor cells that allowed expansion of MDSCs, cytokines in Hepa1-6 cell culture medium and murine serum were examined using a cytokine array. Stem cell factor (SCF) was implicated as the relevant cytokine. When recombinant SCF was added to the spleen cell culture medium, MDSC expansion could occur. In the presence of c-kit blockade, this effect of SCF was partially reversed. In conclusion, MDSCs can be expanded in tumor cells in a process that involves SCF released by tumor cells.

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBPRegulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.

Down-regulation of IDH2 sensitizes cancer cells to erastin-induced ferroptosis

Ferroptosis is a form of regulated cell death induced by lipid peroxidation that is dependent on iron. This pathway is being considered as an alternative anticancer therapeutic strategy, and the chemoreagent erastin induces ferroptosis by blocking system Xc−, which causes a cysteine shortage that depletes intracellular GSH. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is major enzyme that produces NADPH, which is a crucial source for mitochondrial GSH turnover. Therefore, we hypothesized that down-regulation of IDH2 would have a synergic effect on erastin-induced ferroptosis. Here, we investigated the effect of IDH2 knockdown on ferroptosis in human HT1080 fibrosarcoma and murine Hepa1-6 hepatoma cells cultured in vitro as well as in an in vivo model of allografted Hepa1-6 cells in nude mice. Our results show that susceptibility to ferroptosis was substantially increased when IDH2 was down-regulated. This study supports that IDH2 has protective effect against ferroptotic cell death, and that the enzyme could be targeted to sensitize cancer cells to ferroptosis.

Involvement of aryl hydrocarbon receptor in the cytotoxicity of corannulene and its derivatives

Despite numerous studies on the toxicities of planar polycyclic aromatic hydrocarbons (PAHs), very little is known about the toxicological profiles of non-planar PAHs. In the present study, the cytotoxicity of corannulene (COR), a typical bowl-shaped PAH with a myriad of applications in the area of material chemistry, and benzo[a]pyrene (BaP), a typical planar PAH with similar molecular weight, were systematically compared in various cell lines. Compared with BaP, exposure to COR resulted in less cytotoxic responses in both human (HepG2) and murine (Hepa1-6) hepatoma cells, which was characterized with a slower cellular accumulation as well as a weaker induction of cytochrome P450 1 (CYP1/Cyp1) isozymes. Knockdown of aryl hydrocarbon receptor (AhR) by siRNA attenuated the inductive effect of COR on CYP1A/Cyp1a mRNA levels in these two cell lines. Further analysis revealed that derivatization greatly influenced the cytotoxicity of COR, which was positively correlated with their binding affinities to the AhR, as demonstrated by in silico molecular docking. Overall, these results suggest that AhR appears to be involved in the cytotoxic responses of COR and its derivatives, providing a fundamental understanding of the biological effects of bowl-like PAHs.

Crebl2 regulates cell metabolism in muscle and liver cells

We previously identified Drosophila REPTOR and REPTOR-BP as transcription factors downstream of mTORC1 that play an important role in regulating organismal metabolism. We study here the mammalian ortholog of REPTOR-BP, Crebl2. We find that Crebl2 mediates part of the transcriptional induction caused by mTORC1 inhibition. In C2C12 myoblasts, Crebl2 knockdown leads to elevated glucose uptake, elevated glycolysis as observed by lactate secretion, and elevated triglyceride biosynthesis. In Hepa1-6 hepatoma cells, Crebl2 knockdown also leads to elevated triglyceride levels. In sum, this works identifies Crebl2 as a regulator of cellular metabolism that can link nutrient sensing via mTORC1 to the metabolic response of cells.

The nuclear receptor Shp regulates morphine withdrawal syndrome via modulation of Ugt2b expression in mice

In this study, we aimed to investigate a potential role of small heterodimer partner (Shp, a nuclear receptor) in regulation of morphine withdrawal syndrome and to determine the mechanisms thereof. Somatic opiate withdrawal and pharmacokinetic experiments were performed with wild-type (WT) and Shp knockout (Shp-KO) mice. Regulatory effects of Shp on Ugt2b expression were assessed in vitro (using mouse hepatoma Hepa1-6 cells) and in vivo (using Shp-KO mice). Ugt2b mRNA and protein expressions were determined by qPCR and Western blotting, respectively. Microsomal Ugt2b activity was measured with morphine and chloramphenicol. Luciferase reporter, promoter analysis and chromatin immunoprecipitation assays were performed to identify the Hnf1α- and Rev-erbα-binding sites in Ugt2b36 promoter. Protein-protein interactions were explored using co-immunoprecipitation assays. Shp ablation exacerbated morphine withdrawal syndrome in mice. Furthermore, systemic and liver exposures of morphine were elevated in Shp-KO mice due to reduced metabolism. Down-regulation of morphine metabolism was supported by down-regulated expressions of Ugt2b genes in Shp-KO mice. Regulation of Ugt2b genes by Shp was confirmed in mouse hepatoma Hepa1-6 cells. Moreover, Shp positively regulated Ugt2b36 expression through repression of Dec2 and Rev-erbα, two negative regulators of Ugt2b36 enzyme. Rev-erbα repressed Ugt2b36 transcription via direct binding to a specific response element (located at −30/−15 bp) in promoter region of Ugt2b36, whereas Dec2 acted on Ugt2b36 expression via suppression of Hnf1α-transactivation of Ugt2b36 gene. In conclusion, Shp regulated morphine withdrawal syndrome via modulation of Ugt2b expression and detoxification capacity. Targeting Shp may represent a novel approach for management of morphine dependence.

Mechanisms of G0/G1 phase arrest induced by longevity-assurance homologue 2 in Hepa1-6 hepatoma cells

Objective To investigate the effects of longevity-assurance homologue gene 2 (Lass2) on the cell cycle ofmouse hepatic carcinoma cell line Hepa1-6 and the related mechanism. Methods Lass2 recombinant eukaryotic expression vector has been successfully constructed in the early stage, and were transfected in Hepa1-6 hepatoma cell line 48 h by liposome 2000. The cell cycle of each group (negative control, empty load, experimental group) was detected by flow cytometry (PI). The relative expression levels of Lass2, Cyclin D1, nuclear factor-κB (NF-κB) p65 mRNA and protein were detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting; In order to further verify and clarify the mechanism, the C57BL/6J mouse hepatic carcinoma in situ model was constructed by injecting Hepa1-6 hepatic carcinoma cell suspension directly into the liver parenchyma of C57BL/6J mice. The naked plasmid was injected into the tail vein separately by hydrodynamics, and the total RNA and total protein in liver tissue of each group were extracted after transfection. The relative expression levels of Lass2, Cyclin D1, NF-κB p65 mRNA and protein in mice liver were detected by FQ-PCR and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting. Results The relative expression levels of Lass2 mRNA and protein in Hepa1-6 hepatoma cells were significantly higher than those in negative control group and blank control group (F=1 486.895, all P=0.000; protein: F=55.964, all P=0.000), and the percentage of cell cycle G0/G1 phase increased significantly (F=239.867, all P=0.000), significantly decreased in G2/M phase and S phase (FG2/M=13.462, PG2/M=0.003, 0.005; FS=46.207, all P=0.000), at the same time, the levels of NF-κB p65, CyclinD1 mRNA and protein in the experimental group were decreased (mRNA: FNF-κB p65=29.993, FCyclin D1=113.260, all P=0.000; protein: FNF-κB p65=17.679, P=0.003, 0.002; FCyclin D1=176.357, all P=0.000), the phosphorylation of NF-κB p65 were significantly downregulated (FNF-κB p-p65=33.817, P=0.000). Conclusion Lass2 gene blocks the cell cycle of Hepa1-6 cells in G0/G1 phase, and its mechanism may be related to inhibits NF-κB p65 activation and expression and down-regulation of Cyclin D1 transcription and protein expression. Key words: Longevity-assurance homologue gene 2; Hepatocellular carcinoma; Cell cycle; Nuclear factor-κB p65; Cyclin D1

A negative feedback loop between microRNA-378 and Nrf1 promotes the development of hepatosteatosis in mice treated with a high fat diet

BackgroundsThe incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing due to the prevalence of obesity. NAFLD is a major risk factor of hepatocellular carcinoma (HCC). Even with successful surgical removal, the presence of NAFLD is associated with an increased recurrence of HCC. Despite the extensive study of NAFLD, its underlying mechanism(s) remains essentially unknown and there are no FDA-approved drugs for its treatment. Alterations in microRNA (miR) expression have been observed in human fatty livers. However, regulatory mechanism(s) of miRNA biogenesis and their role in regulating the development of NAFLD is poorly described. MethodsWe used immunohistochemistry, luciferase assays and immunoblotting to study the regulatory mechanism of miR-378 biogenesis. Wild-type mice kept on a high fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378 to study loss and gain-of functions of miR-378. ResultsmiR-378 was significantly increased in fatty livers of dietary obese mice and human hepatoma HepG2 cells with accumulated lipid. Further studies identified NRF1 (Nuclear receptor factor 1), a key regulator of fatty acid oxidation (FAO), as a direct target of miR-378. Overexpression of miR-378 impaired FAO and promoted lipid accumulation in murine hepatoma Hepa1-6 cells. In contrast, knockdown of miR-378 using its ASO (anti-sense oligo) improved FAO and reduced intracellular lipid content in Hepa1-6 cells. Liver-specific expression of miR-378 impaired FAO, which subsequently promoted the development of hepatosteatosis. Antagonizing miR-378 via injecting miR-378-ASO into HFD-treated mice led to increased expression of Nrf1, improved FAO and decreased hepatosteatosis. Additional knockdown of up-regulated Nrf1 offset the effects of miR-378-ASO, suggesting that Nrf1 mediated the inhibitory effect of miR-378-ASO on hepatosteatosis. Furthermore, Nrf1 was identified as a transcriptional repressor of miR-378. Ablation of Nrf1 using its shRNA in livers led to increased miR-378, which subsequently resulted in reduced FAO and elevated hepatic lipid content. ConclusionsThese findings identified a negative feedback loop between miR-378 and Nrf1 that promotes the pathogenesis of hepatosteatosis, and suggests the use of miR-378 as a potential therapeutic target for NAFLD.

Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage.

Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

The aryl hydrocarbon receptor is required for induction of p21cip1/waf1 expression and growth inhibition by SU5416 in hepatoma cells.

The aryl hydrocarbon receptor (AhR) is a potential clinical target for cancer and autoimmune dysfunction. Identifying selective AhR modulators that produce desirable clinical outcomes represents an opportunity for developing new anti-cancer agents. Repurposing clinically-used drugs with established safety profiles that activate the AhR represents a good starting place to pursue this goal. In this study, we characterized the AhR-dependent effects of SU5416 (Semaxanib) following its identification in a small-molecule library screen. SU5416 potently activated AhR-dependent reporter genes, induced AhR nuclear localization, facilitated AhR-DNA binding, and increased, expression of its endogenous target genes. SU5416 significantly inhibited proliferation of Hepa1 hepatoma cells in an AhR-dependent manner, but did not induce apoptosis. SU5416 also inhibited the growth of human HepG2 liver cancer cells. The effects of SU5416 correlated with an increased G1 population and increased expression of cell cycle inhibitor p21cip1/waf1 at both the mRNA and protein level. Increased expression of p21cip1/waf1 by SU5416 required expression of both AhR and Arnt. In addition, evidence for long-term activation of the AhR in vivo by a single dose of SU5416 was identified by analyzing published microarray data. Our results provide support for continued investigation of the AhR as therapeutic for cancers such as hepatocellular carcinoma. In addition, our findings raise the possibility that some of the previously observed anti-proliferative effects of SU5416 may be due to activation of the AhR.

Reduced SHARPIN and LUBAC Formation May Contribute to CCl₄- or Acetaminophen-Induced Liver Cirrhosis in Mice.

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl4) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.