JCAD deficiency delayed liver regenerative repair through the Hippo-YAP signalling pathway.

BACKGROUND AND AIMS: Liver regeneration retardation post partial hepatectomy (PH) is a common clinical problem after liver transplantation. Identification of key regulators in liver regeneration post PH may be beneficial for clinically improving the prognosis of patients after liver transplantation. This study aimed to clarify the function of junctional protein-associated with coronary artery disease (JCAD) in liver regeneration post PH and to reveal the underlying mechanisms. METHODS: JCAD knockout (JCAD-KO), liver-specific JCAD-KO (Jcad△Hep) mice and their control group were subjected to 70% PH. RNA sequencing was conducted to unravel the related signalling pathways. Primary hepatocytes from KO mice were treated with epidermal growth factor (EGF) to evaluate DNA replication. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) live-imaging system was used to visualise the phases of cell cycle. RESULTS: Both global and liver-specific JCAD deficiency postponed liver regeneration after PH as indicated by reduced gene expression of cell cycle transition and DNA replication. Prolonged retention in G1 phase and failure to transition over the cell cycle checkpoint in JCAD-KO cell line was indicated by a FUCCI live-imaging system as well as pharmacologic blockage. JCAD replenishment by adenovirus reversed the impaired DNA synthesis in JCAD-KO primary hepatocyte in exposure to EGF, which was abrogated by a Yes-associated protein (YAP) inhibitor, verteporfin. Mechanistically, JCAD competed with large tumour suppressor 2 (LATS2) for WWC1 interaction, leading to LATS2 inhibition and thereafter YAP activation, and enhanced expression of cell cycle-associated genes. CONCLUSION: JCAD deficiency led to delayed regeneration after PH as a result of blockage in cell cycle progression through the Hippo-YAP signalling pathway. These findings uncovered novel functions of JCAD and suggested a potential strategy for improving graft growth and function post liver transplantation. KEY POINTS: JCAD deficiency leads to an impaired liver growth after PH due to cell division blockage. JCAD competes with LATS2 for WWC1 interaction, resulting in LATS2 inhibition, YAP activation and enhanced expression of cell cycle-associated genes. Delineation of JCADHippoYAP signalling pathway would facilitate to improve prognosis of acute liver failure and graft growth in living-donor liver transplantation.

TGF-ß1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-ß/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

JCAD deficiency attenuates activation of hepatic stellate cells and cholestatic fibrosis.

BACKGROUND/AIMS: Cholestatic liver diseases including primary biliary cholangitis (PBC) are associated with active hepatic fibrogenesis, which ultimately progresses to cirrhosis. Activated hepatic stellate cells (HSCs) are the main fibrogenic effectors in response to cholangiocyte damage. JCAD regulates cell proliferation and malignant transformation in nonalcoholic steatoheaptitis-associated hepatocellular carcinoma (NASH-HCC). However, its participation in cholestatic fibrosis has not been explored yet. METHODS: Serial sections of liver tissue of PBC patients were stained with immunofluorescence. Hepatic fibrosis was induced by bile duct ligation (BDL) in wild-type (WT), global JCAD knockout mice (JCAD-KO) and HSC-specific JCAD knockout mice (HSC-JCAD-KO), and evaluated by histopathology and biochemical tests. In situ-activated HSCs isolated from BDL mice were used to determine effects of JCAD on HSC activation. RESULTS: In consistence with staining of liver sections from PBC patients, immunofluorescent staining revealed that JCAD expression was identified in smooth muscle α-actin (α-SMA)-positive fibroblast-like cells and was significantly up-regulated in WT mice with BDL. JCAD deficiency remarkably ameliorated BDL-induced hepatic injury and fibrosis, as documented by liver hydroxyproline content, when compared to WT mice with BDL. Histopathologically, collagen deposition was dramatically reduced in both JCAD-KO and HSC-JCAD-KO mice compared to WT mice, as visualized by Trichrome staining and semi-quantitative scores. Moreover, JCAD deprivation significantly attenuated in situ HSC activation and reduced expression of fibrotic genes after BDL. CONCLUSION: JCAD deficiency effectively suppressed hepatic fibrosis induced by BDL in mice, and the underlying mechanisms are largely through suppressed Hippo-YAP signaling activity in HSCs.

Association of KRAS, NRAS, BRAF and PIK3CA gene mutations with clinicopathological features, prognosis and ring finger protein 215 expression in patients with colorectal cancer.

The relationships of KRAS, NRAS, BRAF and PIK3CA gene mutations with the clinicopathological features and prognosis of colorectal cancer (CRC) in patient are lacking. Furthermore, the role of ring finger protein 215 (RNF215) in CRC patients with KRAS, NRAS, BRAF and PIK3CA mutations remains unclear. In the present study, 182 surgical resection specimens from patients with primary CRC for retrospective analysis, were collected. KRAS/NRAS/BRAF/PIK3CA gene mutations were confirmed by an amplification-refractory mutation system. Immunohistochemistry (IHC) was conducted to confirm KRAS, NRAS, BRAF and PIK3CA protein expression. RNF215 expression in patients with CRC was evaluated using TIMER 2.0 database and IHC. The individual mutation rates of KRAS, NRAS, BRAF and PIK3CA were 40.7% (74/182), 4.4% (8/182), 4.4% (8/182) and 3.3% (6/182), respectively. The KRAS exon 2 mutation rate was the highest (61.5%, 64/104), and these mutations mainly occurred at codons 12 and 13. KRAS/NRAS/BRAF/PIK3CA wild-type CRC patients had significantly longer overall survival and disease-free survival than mutated KRAS/NRAS/BRAF/PIK3CA CRC patients (P<0.05). Overall, 45.4% (5/11) of patients with PIK3CA mutations had concomitant KRAS mutations. The KRAS/NRAS/BRAF/PIK3CA gene mutation rate in patients with lymph node metastasis (76.1%, 35/46) was significantly higher than that in patients without lymph node metastasis (50.8%, 69/136) (P=0.0027). There were no significant differences in IHC expression between patients with and without KRAS, NRAS, BRAF and PIK3CA mutations (P>0.05). The TIMER 2.0 analysis showed that RNF215 expression was significantly higher in the mutated BRAF group than in the wild-type BRAF group in CRC (P<0.05). In conclusion, KRAS is the most commonly mutated gene, and KRAS mutations may be a poor prognostic factor for patients with CRC. KRAS wild-type patient resistance may be related to PIK3CA gene mutations, although this needs further verification in larger cohorts. BRAF mutations may be associated with RNF215 expression in patients with CRC.

Ring finger protein 215 is a potential prognostic biomarker involved in immune infiltration and angiogenesis in colorectal cancer.

The prognostic value of ring finger protein 215 (RNF215) in colorectal cancer (CRC) is unclear. Herein, the present study aimed to investigate the precise value of RNF215 based on CRC datasets from The Cancer Genome Atlas (TCGA) and clinical cases. CRC patient data was collected from TCGA and clinical samples from the Department of Pathology, Shanghai Fifth People's Hospital, Fudan University (Shanghai, China). Logistic regression analysis was used to investigate the correlations between RNF215 and clinicopathological characteristics. The predictive value of RNF215 for the clinical outcome of CRC was determined using Kaplan-Meier curves and Cox regression. Gene set enrichment analysis (GSEA), single-sample GSEA (ssGSEA), and angiogenesis analysis were also conducted to investigate the biological role of RNF215. Immunohistochemistry was conducted to validate the results. The results of the present study confirmed that RNF215 protein expression was significantly associated with age, lymphatic invasion, and overall survival (OS). Univariate analysis showed that upregulation of RNF215 in CRC was significantly associated with age and lymphatic invasion. Kaplan-Meier survival analysis revealed that high RNF215 expression predicted poorer OS and disease-specific survival. A total of nine experimentally detected RNF215-binding proteins were identified with the STRING tool and Cytoscape software. GSEA suggested that RNF215 was associated with several important pathways involved in tumor occurrence, including the Kyoto Encyclopedia of Genes and Genomes MAPK signaling pathway and the WikiPathway RAS signaling pathway. ssGSEA confirmed that RNF215 was significantly expressed in natural killer cells, CD8 T cells and T helper cells. Angiogenesis analysis revealed that numerous angiogenesis-related genes had the same expression trend as RNF215 in CRC. The immunostaining results indicated that RNF215 expression was significantly higher in CRC tissues than in corresponding normal tissues. In conclusion, increased RNF215 expression may be a potential molecular marker predictive of poor survival and a treatment target in CRC. In addition, RNF215 may participate in the formation of CRC through a variety of signaling pathways.

METTL16-mediated translation of CIDEA promotes non-alcoholic fatty liver disease progression via m6A-dependent manner.

Background: As the most prevalent chemical modifications on eukaryotic mRNAs, N6-methyladenosine (m6A) methylation was reported to participate in the regulation of various metabolic diseases. This study aimed to investigate the roles of m6A methylation and methyltransferase-like16 (METTL16) in non-alcoholic fatty liver disease (NAFLD). Methods: In this study, we used a model of diet-induced NAFLD, maintaining six male C57BL/6J mice on high-fat diet (HFD) to generate hepatic steatosis. The high-throughput sequencing and RNA sequencing were performed to identify the m6A methylation patterns and differentially expressed mRNAs in HFD mice livers. Furthermore, we detected the expression levels of m6A modify enzymes by qRT-PCR in liver tissues, and further investigated the potential role of METTL16 in NAFLD through constructing overexpression and a knockdown model of METTL16 in HepG2 cells. Results: In total, we confirmed 15,999 m6A recurrent peaks in HFD mice and 12,322 in the control. Genes with differentially methylated m6A peaks were significantly associated with the dysregulated glucolipid metabolism and aggravated hepatic inflammatory response. In addition, we identified five genes (CIDEA, THRSP, OSBPL3, GDF15 and LGALS1) that played important roles in NAFLD progression after analyzing the differentially expressed genes containing differentially methylated m6A peaks. Intriguingly, we found that the expression levels of METTL16 were substantially increased in the NAFLD model in vivo and in vitro, and further confirmed that METTL16 upregulated the expression level of lipogenic genes CIDEA in HepG2 cells. Conclusions: These results indicate the critical roles of m6A methylation and METTL16 in HFD-induced mice and cell NAFLD models, which broaden people's perspectives on potential m6A-related treatments and biomarkers for NAFLD.

microRNA-106b-5p Promotes Cell Growth and Sensitizes Chemosensitivity to Sorafenib by Targeting the BTG3/Bcl-xL/p27 Signaling Pathway in Hepatocellular Carcinoma.

microRNAs (miRNAs) and miRNA-mediated regulatory networks are promising candidates in the prevention and treatment of cancer, but the role of specific miRNAs involved in hepatocellular carcinoma (HCC) remains to be elusive. Herein, we found that miR-106b-5p is upregulated in both HCC patients' tumor tissues and HCC cell lines. The miR-106b-5p expression level was positively correlated with α-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), and tumor size. Overexpression of miR-106b-5p promoted cell proliferation, migration, cell cycle G1/S transition, and tumor growth, while decreased miR-106b-5p expression had opposite effects. Mechanistic studies showed that B-cell translocation gene 3 (BTG3), a known antiproliferative protein, was a direct target of miR-106b-5p, whose expression level is inversely correlated with miR-106b-5p expression. Moreover, miR-106b-5p positively regulates cell proliferation in a BTG3-dependent manner, resulting in upregulation of Bcl-xL, cyclin E1, and CDK2, as well as downregulation of p27. More importantly, we also demonstrated that miR-106b-5p enhances the resistance to sorafenib treatment in a BTG3-dependent manner. The in vivo findings showed that mice treated with a miR-106b-5p sponge presented a smaller tumor burden than controls, while the mice injected cells treated with miR-106b-5p had more considerable tumor burden than controls. Altogether, these data suggest that miR-106b-5p promotes cell proliferation and cell cycle and increases HCC cells' resistance to sorafenib through the BTG3/Bcl-xL/p27 signaling pathway.

Anti-viral activity of Staphylococcus aureus lysates against herpes simplex virus type-I infection: an in vitro and in vivo study.

AIM: To investigate the effect of Staphylococcus aureus (S. aures) lysates (SALs) on herpes simplex virus type-I (HSV1) infection in human corneal epithelial (HCE) cells and in a mouse model of HSV1 keratitis. METHODS: HCE, Vero, HeLa, and BV2 cells were infected with HSV1 [HSV1 f strain, HSV1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV1g]. Pre- or post-infection, SAL at various concentrations was added to the culture medium for 24h. GFP fluorescence in HSV1g or plaque formation by HSV1f were examined. The effects of heat-treated SAL, precooled acetone-precipitated SAL, and SAL subjected to ultrafiltration (100 kDa) were evaluated. The effects of other bacterial components and lysates on HSV1 infection were also tested, including lipoteichoic acid (LTA), peptidoglycan (PGN), staphylococcal protein A (SPA), and α-hemolysin from S. aureus (α-toxin) as well as lysates from a wild-type S. aureus strain, S. epidermidis, and Escherichia coli (W-SAL, SEL, and ECL, respectively). In addition, SAL eye drops were applied topically to BALB/c mice with HSV1 keratitis, followed by in vivo observations. RESULTS: The cytopathic effect, plaque formation (HSV1f), and GFP expression (HSV1g) in infected cells were inhibited by SAL in a dose-dependent manner. The active component of SAL (≥100 kDa) was heat-sensitive and retained activity after acetone precipitation. In HSV1g-infected cells, treatment with LTA-sa, α-toxin, PGN-sa, or SPA did not inhibit GFP expression. SAL, W-SAL, and SEL (but not ECL) decreased GFP expression. In mice with HSV1 keratitis, SAL reduced corneal lesions by 71%. CONCLUSION: The results of this study demonstrate that SAL can be used to inhibit HSV1 infection, particularly keratitis. Further studies are needed to determine the active components and mechanism underlying the effects of SAL.

Prognostic and Immunological Role of THBS2 in Colorectal cancer.

OBJECTIVE: Thrombospondin 2 (THBS2) acts as oncogenic or tumor suppressive gene in diverse cancers. Here we studied the prognostic and immunological role of THBS2 in colorectal cancer (CRC) using bioinformatic analysis. METHODS: The genetic and protein expression of THBS2 in CRC were explored across several databases, including ONCOMINE, GEPIA2, TIMER 2.0, UALCAN and HPA databases. Correlation between THBS2 expression and clinical features in CRC was assessed using UALCAN tool. Prognostic analysis was performed using GEPIA2 and PrognoScan. Immune infiltration correlation with THBS2 in CRC was investigated with TIMER 2.0 and TISIDB. THBS2 binding and correlated genes were analyzed using String, GEPIA2, and TIMER 2.0. RESULTS: THBS2 was significantly higher in CRC across multiple databases. Age and histological subtype were correlated with THBS2 level. High THBS2 expression correlated with short overall and disease-free survival. THBS2 expression was positively correlated with immune infiltrates in CRC. Moreover, extracellular matrix structural constituent and organization, PI3K-Akt pathway, were involved in the functional mechanisms of THBS2. CONCLUSIONS: THBS2 correlates with poor prognosis and immune infiltration in CRC. THBS2 may act as a prognostic and immunological biomarker for CRC.

An aberrant F8 intron 1 inversion with concomitant large duplication and deletion in a Chinese severe hemophilia A patient.

OBJECTIVES: Hemophilia A (HA, OMIM: 306700) is an X-linked recessive bleeding disorder, caused by defects of the F8 gene which encodes the coagulation factor VIII (FVIII). F8 intron 22 and intron 1 inversion (Inv22 and Inv1) account for ∼45% and 1-5% of severe HA cases, respectively. We herein described an aberrant Inv1 with concomitant large duplication and deletion in a Chinese severe HA patient. METHODS: Long distance PCR and multiplex PCR were used to detect Inv22 and Inv1. Multiplex ligation-dependent probe amplification (MLPA) was applied to examine exonic duplication and deletion of the F8 gene. Coverage analysis of read depth data from whole-genome sequencing (WGS) was used to analyze the intronic duplication and deletion of the F8 gene. RESULTS: We have identified an aberrant F8 Inv1 in a 1-year-old Chinese severe HA patient showing inversed int1h-1 and normal int1h-2. Coverage analysis of WGS data further illustrated the aberrant Inv1 with concomitant a duplication of 117 kb and a deletion of 1.8 kb. CONCLUSION: In conclusion, we reported an aberrant Inv1 with concomitant large duplication and deletion in a severe Chinese HA patient. Moreover, WGS provides rapid genetic diagnosis of hereditary disorders with point mutations, deletions, insertions and CNVs.

Hepalatide ameliorated progression of nonalcoholic steatohepatitis in mice.

BACKGROUND: No FDA-approved medications are available for the treatment of nonalcoholic steatohepatitis (NASH). The present study aimed to assess the effects of Hepalatide, a sodium taurocholate cotransporting polypeptide (NTCP) receptor-binding agent, on metabolic and histopathologic changes of a mouse model of NASH caused by high fat/calorie diet plus high fructose/glucose in drinking water (HFCD-HF/G) for 16 weeks. METHODS: Male mice were randomly divided into 4 groups: controls (normal diet), HFCD-HF/G group, HFCD-HF/G plus low or high dose of Hepalatide (20 or 60 mg/kg, LH or HH, s.c. from 9 to 16 weeks). RESULTS: Compared to HFCD-HF/G-fed mice, serum triglyceride and cholesterol levels in mice fed HFCD-HF/G plus LH or HH were decreased. The treatment with Hepalatide decreased serum alanine aminotransferase levels significantly. Liver histology and TUNEL staining showed that Hepalatide remarkably attenuated inflammation, hepatocellular steatosis and apoptosis. Hepalatide treatment decreased fasting blood glucose, serum insulin and HOMA insulin resistance index in the HH group. Moreover, Masson's staining, semi-quantitative score of fibrosis, and hydroxyproline content demonstrated that Hepalatide mitigated fibrotic progression in this murine NASH model. Additionally, most components of liver and few serum bile acids were increased in mice treated with HH. CONCLUSION: Hepalatide effectively alleviated the pathological process, metabolic profile, hepatocellular steatosis and injury, insulin resistance, halted hepatic fibrotic progression in a mouse model of NASH, most likely through the increase of serum bile acids.

Succinate-GPR-91 receptor signalling is responsible for nonalcoholic steatohepatitis-associated fibrosis: Effects of DHA supplementation.

BACKGROUND AND AIMS: Treatment of non-alcoholic steatohepatitis (NASH) is challenging, because suppressing fibrotic progression has not been achieved consistently by drug candidates currently in clinical trials. The aim of this study was to investigate the molecular interplays underlying NASH-associated fibrosis in a mouse NASH model and human specimens. METHODS: Mice were divided into 4 groups: Controls; NASH (high fat/Calorie diet plus high fructose and glucose in drinking water, HFCD-HF/G) for 16 weeks; HFCD-HF/G plus docosahexaenoic acid (DHA) for 16 or 8 weeks. RESULTS: Along with NASH progression, fibrotic deposition was documented in HFCD-HF/G-fed mice. Liver succinate content was significantly increased along with decreased expression of succinate dehydrogenase-A (SDH-A) in these mice; whereas, GPR-91 receptor expression was much enhanced in histology compared to control mice, and co-localized histologically with hepatic stellate cells (HSCs). Succinate content was increased in fatty acid-overloaded primary hepatocytes with significant oxidant stress and lipotoxicity. Exposure to succinate led to up-regulation of GPR-91 receptor in primary and immortalized HSCs. In contrast, suppression of GPR-91 receptor expression abolished succinate stimulatory role in GPR-91 expression and extracellular matrix production in HSCs. All these changes were minimized or abrogated by DHA supplementation in vivo or in vitro. Moreover, GPR-91 receptor expression correlates with severity of fibrosis in human NASH biopsy specimens. CONCLUSION: Succinate accumulation in steatotoic hepatocytes may result in HSC activation through GPR-91 receptor signalling in NASH progression, and the cross-talk between hepatocytes and HSC through GPR-91 signalling is most likely to be the molecular basis of fibrogenesis in NASH.

Characterization of a murine nonalcoholic steatohepatitis model induced by high fat high calorie diet plus fructose and glucose in drinking water.

There are varieties of murine models of nonalcoholic steatohepatitis (NASH) with different pathophysiologic characteristics. For preclinical assessment, a standardized model would allow comparisons of various pharmacotherapeutic candidates in efficacy, pharmacokinetics, pharmaco-metabolism, and adverse effects under a same system. The present study aims to characterize murine NASH models by comparing end-points of major abnormalities. NASH was induced by feeding high fructose/glucose in drinking water (HF/G), high-fat/calorie diet (HFCD), and in combination (HFCD-HF/G) in mice for 8 or 16 weeks. HF/G feeding caused a minimal fat accumulation and increase in free fatty acids (FFA). In contrast, HFCD-HF/G feeding resulted in a remarkable increase in body weight, subcutaneous and visceral adipose tissue, macrosteatosis with a nearly seven-fold increase in triglyceride and FFA content, accompanied with marked hepatocellular injury, inflammatory responses, fibrosis, and insulin resistance, and represented as typical NASH in histopathology, metabolic, and adipokine profiles in a progressive manner. Meanwhile, mice fed HFCD displayed significant steatosis, necroptosis, fibrosis, insulin resistance, metabolic, and adipokine profiles, and the extent is less than those fed HFCD-HF/G. Significant MCP-1, CCR-2, and NLRP-1/3 activation were found in mice fed HFCD and HFCD-HF/G for 16 weeks, whereas gene expression of CPT-1 and ACOX-1 was down-regulated in these two groups in comparison to the controls. Nuclear receptors, such as SREBP-1c, FXR, LXR-α, PPAR-α, and PPAR-γ, were strikingly elevated in the HFCD-HF/G group. In conclusion, feeding HFCD-HF/G resulted in a reliable NASH model in mice with remarkable necroptosis, steatosis, fibrosis, and insulin resistance as well as a disordered profile of lipid metabolism and adipokine, and HFCD caused significant NASH features in histopathology and metabolic profiles only at a late stage. Whereas HF/G feeding barely led to minimal fat accumulation, some changes at molecular levels and metabolic disturbance in mice.

Trichostatin A inhibits the activation of Hepatic stellate cells by Increasing C/EBP-α Acetylation in vivo and in vitro.

Reversal of activated hepatic stellate cells (HSCs) to a quiescent state and apoptosis of activated HSCs are key elements in the reversion of hepatic fibrosis. CCAAT/enhancer binding protein α (C/EBP-α) has been shown to inhibit HSC activation and promote its apoptosis. This study aims to investigate how C/EBP-α acetylation affects the fate of activated HSCs. Effects of a histone deacetylation inhibitor trichostatin A (TSA) on HSC activation were evaluated in a mouse model of liver fibrosis caused by carbon tetrachloride (CCl4) intoxication. TSA was found to ameliorate CCl4-induced hepatic fibrosis and improve liver function through increasing the protein level and enhancing C/EBP-α acetylation in the mouse liver. C/EBP-α acetylation was determined in HSC lines in the presence or absence of TSA, and the lysine residue K276 was identified as a main acetylation site in C/EBP-α protein. C/EBP-α acetylation increased its stability and protein level, and inhibited HSC activation. The present study demonstrated that C/EBP-α acetylation increases the protein level by inhibiting its ubiquitination-mediated degradation, and may be involved in the fate of activated HSCs. Use of TSA may confer an option in minimizing hepatic fibrosis by suppressing HSC activation, a key process in the initiation and progression of hepatic fibrosis.

Activation of pluripotent genes in hepatic progenitor cells in the transition of nonalcoholic steatohepatitis to pre-malignant lesions.

Nonalcoholic steatohepatitis is considered as a precancerous condition. However, hepatic carcinogenesis from NASH is poorly understood. This study aims to investigate the activation of pluripotent genes (c-Myc, Oct-4, KLF-4, and Nanog) and morphogenic gene (Gli-1) in hepatic progenitor cells from patient specimens and in an animal model to determine the possibility of normal stem/progenitor cells becoming the origin of NASH-HCC. In this study, expression of pluripotent and morphogenic genes in human NASH-HCC tissues was significantly upregulated compared to adjacent non-tumor liver tissues. After feeding high-fat/calorie diet plus high fructose/glucose in drinking water (HFC diet plus HF/G) for up to 12 months, mice developed obesity, insulin resistance, and steatohepatitis with significant necroptotic inflammation and fibrotic progression, as well as occurrence of hyperplastic nodules with dysplasia; and this model represents pathohistologically as a transition from NASH to NASH-HCC in a pre-carcinomatous stage. High expression of pluripotent and morphogenic genes was immunohistochemically visualized in the dysplasia areas of mouse liver, where there were many OV-6-positive cells, indicating proliferation of HOCs in NASH with fibrotic progression. Moreover, oncogenic transcription factors (c-Myc, KLF-4, and Nanog) were co-localized in these hepatic progenitor cells. In conclusion, pluripotent and morphogenic genes may contribute to the reprogramming of hepatic progenitor cells in driving these cells to be the origin of NASH-HCC in a steatotic and inflamed microenvironment.

TSA increases C/EBP­α expression by increasing its lysine acetylation in hepatic stellate cells.

CCAAT enhancer binding protein­α (C/EBP­α) is a transcription factor expressed only in certain tissues, including the liver. It has been previously demonstrated that C/EBP­α may induce apoptosis in hepatic stellate cells (HSCs), raising the question of whether acetylation of C/EBP­α is associated with HSCs, and the potential associated mechanism. A total of three histone deacetylase inhibitors (HDACIs), including trichostatin A (TSA), suberoylanilide hydroxamic acid and nicotinamide, were selected to determine whether acetylation affects C/EBP­α expression. A Cell Counting Kit­8 assay was used to determine the rate of proliferation inhibition following treatment with varying doses of the three HDACIs in HSC­T6 and BRL­3A cells. Western blot analysis was used to examine Caspase­3, ­8, ­9, and ­12 levels in HSC­T6 cells treated with adenoviral­C/EBP­α and/or TSA. Following treatment with TSA, a combination of reverse transcription­quantitative polymerase chain reaction and western blot analyses was used to determine the inherent C/EBP­α mRNA and protein levels in HSC­T6 cells at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. Nuclear and cytoplasmic proteins were extracted to examine C/EBP­α distribution. Co­immunoprecipitation analysis was used to examine the lysine acetylation of C/EBP­α. It was observed that TSA inhibited the proliferation of HSC­T6 cells to a greater extent compared with BRL­3A cells, following treatment with the three HDACIs. TSA induced apoptosis in HSC­T6 cells and enhanced the expression of C/EBP­α. Following treatment of HSC­T6 cells with TSA, inherent C/EBP­α expression increased in a time­dependent manner, and its lysine acetylation simultaneously increased. Therefore, the results of the present study suggested that TSA may increase C/EBP­α expression by increasing its lysine acetylation in HSCs.

Neuroprotective Effect of Salvianolic Acids against Cerebral Ischemia/Reperfusion Injury.

This study investigated the neuroprotective effect of salvianolic acids (SA) against ischemia/reperfusion (I/R) injury, and explored whether the neuroprotection was dependent on mitochondrial connexin43 (mtCx43) via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In vitro, we measured astrocyte apoptosis, mitochondrial membrane potential, and also evaluated the morphology of astrocyte mitochondria with transmission electron microscopy. In vivo, we determined the cerebral infarction volume and measured superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Additionally, mtCx43, p-mtCx43, AKT, and p-AKT levels were determined. In vitro, we found that I/R injury induced apoptosis, decreased cell mitochondrial membrane potential (MMP), and damaged mitochondrial morphology in astrocytes. In vivo, we found that I/R injury resulted in a large cerebral infarction, decreased SOD activity, and increased MDA expression. Additionally, I/R injury reduced both the p-mtCx43/mtCx43 and p-AKT/AKT ratios. We reported that both in vivo and in vitro, SA ameliorated the detrimental outcomes of the I/R. Interestingly, co-administering an inhibitor of the PI3K/AKT pathway blunted the effects of SA. SA represents a potential treatment option for cerebral infarction by up-regulating mtCx43 through the PI3K/AKT pathway.

Mechanism of Mitochondrial Connexin43's Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury.

We observed mitochondrial connexin43 (mtCx43) expression under cerebral ischemia-reperfusion (I/R) injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO). Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. MtCx43, p-mtCx43, protein kinase C (PKC), and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX) and diazoxide (DZX) groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD) groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA) reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

MicroRNA-320a inhibits breast cancer metastasis by targeting metadherin.

Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro, while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3'-untranslated region luciferase of metadherin (MTDH), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo. Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.