Search

Supplementary Information

Figure S5, A, Western blots analysis of IDOs, AHR, PD-L1 and CD86 protein expression in various hepatoma cells. CNL: Chang normal liver cells. B, The Kaplan-Meier survival curve analysis between HCC patients with low and high expression of CD80.

The intestine-specific homeobox transcription factor intestine-specific homeobox (ISX) is an IL6-inducible proto-oncogene implicated in the development of hepatocellular carcinoma, but its mechanistic contributions to this process are undefined. In this study, we provide evidence that ISX mediates a positive feedback loop integrating inflammation, tryptophan catabolism, and immune suppression. We found that ISX-mediated IL6-induced expression of the tryptophan catabolic enzymes Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase in hepatocellular carcinoma cells, resulting in an ISX-dependent increase in the tryptophan catabolite kynurenine and its receptor aryl hydrocarbon receptor (AHR). Activation of this kynurenine/AHR signaling axis acted through a positive feedback mechanism to increase ISX expression and enhance cellular proliferation and tumorigenic potential. RNAi-mediated attenuation of ISX or AHR reversed these effects. In an IDO1-dependent manner, ectopic expression of ISX induced expression of genes encoding the critical immune modulators CD86 (B7-2) and programmed death ligand-1 (PD-L1), through which ISX conferred a significant suppressive effect on the CD8+ T-cell response. In hepatocellular carcinoma specimens, expression of IDO1, kynurenine, AHR, and PD-L1 correlated negatively with survival. Overall, our results identified a feed-forward mechanism of immune suppression in hepatocellular carcinoma organized by ISX, which involves kynurenine-AHR signaling and PD-L1, offering insights into immune escape by hepatocellular carcinoma, which may improve its therapeutic management. Cancer Res; 77(15); 4065–77. ©2017 AACR.

The intestine-specific homeobox transcription factor intestine-specific homeobox (ISX) is an IL6-inducible proto-oncogene implicated in the development of hepatocellular carcinoma, but its mechanistic contributions to this process are undefined. In this study, we provide evidence that ISX mediates a positive feedback loop integrating inflammation, tryptophan catabolism, and immune suppression. We found that ISX-mediated IL6-induced expression of the tryptophan catabolic enzymes Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase in hepatocellular carcinoma cells, resulting in an ISX-dependent increase in the tryptophan catabolite kynurenine and its receptor aryl hydrocarbon receptor (AHR). Activation of this kynurenine/AHR signaling axis acted through a positive feedback mechanism to increase ISX expression and enhance cellular proliferation and tumorigenic potential. RNAi-mediated attenuation of ISX or AHR reversed these effects. In an IDO1-dependent manner, ectopic expression of ISX induced expression of genes encoding the critical immune modulators CD86 (B7-2) and programmed death ligand-1 (PD-L1), through which ISX conferred a significant suppressive effect on the CD8+ T-cell response. In hepatocellular carcinoma specimens, expression of IDO1, kynurenine, AHR, and PD-L1 correlated negatively with survival. Overall, our results identified a feed-forward mechanism of immune suppression in hepatocellular carcinoma organized by ISX, which involves kynurenine-AHR signaling and PD-L1, offering insights into immune escape by hepatocellular carcinoma, which may improve its therapeutic management. Cancer Res; 77(15); 4065–77. ©2017 AACR.

The aryl hydrocarbon receptor (AHR), a major chemical sensor, is thought to play a role in various biological contexts, including cell cycle regulation and tumorigenesis. However, its regulatory mechanisms remain unclear. We propose herein a novel mechanism through which AHR promotes tumorigenesis by targeting expression of the oncogene intestine-specific homeobox (ISX) in hepatocellular carcinoma (HCC). Compared to paired tumor-adjacent tissues and non-HCC tumors, HCCs exhibited an increased and hierarchical pattern of AHR expression. Patients exhibiting high AHR expression had a significantly shorter survival duration, compared to those with low and medium expression. Functionally, AHR was found to target the newly discovered proto-oncogene, ISX, resulting in the increased expression of this gene and its downstream targets, CCND1 and E2F1. Ablation of AHR or ISX in hepatoma cells suppressed cell growth, whereas overexpression promoted cell proliferation and led to enhanced tumorigenic activity in vitro and in vivo. These results provide evidence to support a critical role for the AHR/ISX axis in HCC tumorigenesis and suggest its potential utility as a new therapeutic and prognostic target for HCC.

Transformation of macrophages to foam cells contributes to atherosclerosis. Here, we report that let-7g reduces macrophage transformation and alleviates foam cell apoptosis by suppressing both canonical and non-canonical NF-κB pathways. In the canonical pathway, let-7g inhibits phosphorylation of IKKβ and IκB, down-regulates SREBF2 and miR-33a, and up-regulates ABCA1. In the non-canonical pathway, let-7g directly knocks down MEKK1, IKKα and ablates IKKα phosphorylation. Let-7g's effects in macrophages can be almost completely blocked by inactivation of NF-κB signaling, which suggests that let-7g's effects are primarily mediated through the suppression of NF-κB pathways. NF-κB has been reported to directly activate lin28 transcription, and lin28 is a well-known negative regulator for let-7 biogenesis. Therefore, there is negative feedback between NF-κB and let-7g. Additional macrophages-specific NF-κB knockout in the apoE deficiency mice reduces atherosclerotic lesion by 85%. Let-7g also suppresses p53-dependent apoptosis. Altogether, sufficient let-7g levels are important to prevent NF-κB over-activation in macrophages and to prevent atherosclerosis.

Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis and conveys a significant health burden globally. Critical limb ischaemia encompasses the most severe consequence of PAD. Our previous studies indicate that microRNA let‐7g prevents atherosclerosis and improves endothelial functions. This study aimed to investigate whether and how let‐7g therapy may improve blood flow to ischaemic limbs. The present study shows that let‐7g has multiple pro‐angiogenic effects on mouse ischaemic limb model and could be a potential therapeutic agent for PAD. Mice receiving intramuscular injection of let‐7g had more neovascularization, better local perfusion and increased recruitment of endothelial progenitor cells after hindlimb ischaemia. The therapeutic effects of let‐7g's on angiogenesis are mediated by multiple regulatory machinery. First, let‐7g increased expression of vascular endothelial growth factor‐A (VEGF‐A) and VEGF receptor‐2 (VEGFR‐2) through targeting their upstream regulators HIF‐3α and TP53. In addition, let‐7g affected the splicing factor SC35 which subsequently enhanced the alternative splicing of VEGF‐A from the anti‐angiogenic isoform VEGF‐A165b towards the pro‐angiogenic isoform VEGF‐A164a. The pleiotropic effects of let‐7g on angiogenesis imply that let‐7g may possess a therapeutic potential in ischaemic diseases.

Supplemental digital content is available in the text., Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC.

MicroRNAs (miRNA) have been implicated in HCV infection. The present study analyzed the effects of let-7g on HCV infection in vitro, in clinical tissue and serum samples. Here, we show that the expression of let-7g in serum and liver tissue is significantly higher in patients with sustained virologic response (SVR). We show that interferon (IFN)/ribavirin (RBV) induces let-7g expression through p38/AP-1 signaling. Overexpression of let-7g reduced HCV gene or core protein level and inhibited the HCV viral load. The let-7g and IFN/RBV have additively inhibitory effect on HCV replication. These data implicate let-7g as a new therapeutic drug to additively cooperate with IFN/RBV to repress HCV replication. Key messages: let-7g expression is increased in serum and liver tissue of patients with SVR. Interferon/ribavirin induces let-7g expression through p38/AP-1 signaling. Overexpression of let-7g can repress HCV replication. Let-7g additively cooperates with interferon/ribavirin to repress HCV replication. Lin28B silencing can reverse let-7g expression and repress HCV replication.

AIM: To test for the association between genome-wide methylation and myopia in human and mice. METHODS: Long interspersed nucleotide element 1 (LINE-1) methylation levels were used to surrogate genome-wide methylation level. We first tested for the association between high myopia (

Di(2-ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα-positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF-7 (ERα-dependent) and MDA-MB-231 (ERα-independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT-induced formation of reactive oxygen species in ERα-positive MCF-7 cells, but not in ERα-negative MDA-MB-231 cells. DEHP also significantly protected MCF-7 cells against the genotoxicity of CPT. Genome-wide DNA methylation profiling revealed that after 48 hours of exposure to 100 μM DEHP, MCF-7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP-exposed MCF-7 cells is probably mediated by epigenetic changes, especially through Wnt/β-catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT-induced anti-growth of MCF-7 cells. In summary, DEHP exposure induces acquired CPT-resistance in breast cancer cells and epigenetic changes associated with Wnt/β-catenin signaling activation are probably depending on an ER-positive status.

Hepatocellular carcinoma (HCC), the major type of liver cancer, is among the most lethal cancers owing to its aggressive nature and frequently late detection. Therefore, the possibility to identify early diagnostic markers could be of significant benefit. Urine has especially become one of the most attractive body fluids in biomarker discovery as it can be obtained non-invasively in large quantities and is stable as compared with other body fluids. To identify potential protein biomarker for early diagnosis of HCC, we explored protein expression profiles in urine from HCC patients and normal controls (n = 44) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry (nanoLC–MS/MS) and stable isotope dimethyl labeling. We have systematically mapped 91 proteins with differential expressions (p, Highlights • An integrated proteogenomic analysis is applied to identify biomarkers for HCC. • Genomic amplifications of S100A9 and GRN co-occur in tumors from HCC patients. • S100A9 and GRN are co-expressed in tumor and urine samples from HCC patients. • Amplifications of S100A9 and GRN are associated with poor survival of HCC patients.

Metabolic syndrome (MetS) confers increased risks of cardiovascular disease (CVD). Both vitamin D3and adipocytokines (especially adiponectin and leptin) have a great impact on CVD and MetS. In vitamin D3metabolism, the vitamin D325-hydroxylase (CYP27A1) and 25-hydroxyvitamin D31-alpha-hydroxylase (CYP27B1) are two key enzymes. This study aimed to examine the influence of vitamin D3CYP27 single nucleotide polymorphisms (SNPs) on adipocytokines and MetS. Cross-sectional data and DNA samples were collected from male volunteers (n=649, age: 55.7 ± 4.7 years). Two tagging SNPs,CYP27A1 rs4674344andCYP27B1 rs10877012, were selected from the HapMap project. MetS was significantly associated with theCYP27A1 rs4674344SNP(P=0.04)and the ratio of adiponectin/leptin (A/L ratio) was most correlated to theCYP27A1 rs4674344SNP, appearing to be significantly lower in T-carriers than in AA subjects (3.7 ± 4.0 versus 5.1 ± 6.0,P=0.001) and significantly negatively associated after adjustment. For each MetS component associated with theCYP27A1 rs4674344SNP, the A/L ratios were significantly negative in preclinical stage (condition not meeting the individual criteria), except the blood pressure. In conclusion,CYP27A1 rs4674344SNP, A/L ratio, and MetS are significantly associated and T-carriers might have a higher risk of developing MetS due to low A/L ratios in the preclinical stage.

Investors would like to be confident that a PV plant will work as promised before investing large sums of money. Data showing that PV modules can withstand thousands of hours of accelerated testing are a comfort, but are not sufficient to characterize the economic useful life (EUL) of the whole PV system. An inability to be confident of every aspect of the system can motivate less favorable contract terms. In the end, a winning partnership is obtained when a carefully engineered, durable design is married with a robust quality management system and the marriage is extended across all parts of the value chain. However, this marriage is challenging because of the conventional cultural divide between design engineers and quality management specialists. International standards are being developed to facilitate this partnership and include technology-specific requirements in the quality management systems used for component manufacture and system design, construction and operation.

The insulin-like growth factor (IGF)-1 signaling is relevant in regulating cell growth and cytokine secretions by glioblastomas. MicroRNAs determine the cell fate in glioblastomas. However, relationships between IGF-1 signaling and miRNAs in glioblastoma pathogenesis are still unclear. Our aim was to validate the IGF-1-mediated mRNA/miRNA regulatory network in glioblastomas. Using in silico analyses of mRNA array and RNA sequencing data from The Cancer Genome Atlas (TCGA), we identified 32 core enrichment genes that were highly associated with IGF-1-promoted cytokine-cytokine receptor interactions. To investigate the IGF-1-downregulated miRNA signature, microarray-based approaches with IGF-1-treated U87-MG cells and array data in TCGA were used. Four miRNAs, including microRNA (miR)-9-5p, miR-9-3p, miR-181d, and miR-130b, exhibited an inverse correlation with IGF-1 levels. The miR-181d, that targeted the most IGF-1-related cytokine genes, was significantly reduced in IGF-1-treated glioma cells. Statistical models incorporating both high-IGF-1 and low-miR-181d statuses better predicted poor patient survival, and can be used as an independent prognostic factor in glioblastomas. The C-C chemokine receptor type 1 (CCR1) and interleukin (IL)-1b demonstrated inverse correlations with miR-181d levels and associations with patient survival. miR-181d significantly attenuated IGF-1-upregulated CCR1 and IL-1b gene expressions. These findings demonstrate a distinct role for IGF-1 signaling in glioma progression via miR-181d/cytokine networks.

The intestine-specific homeobox transcription factor intestine-specific homeobox (ISX) is an IL6-inducible proto-oncogene implicated in the development of hepatocellular carcinoma, but its mechanistic contributions to this process are undefined. In this study, we provide evidence that ISX mediates a positive feedback loop integrating inflammation, tryptophan catabolism, and immune suppression. We found that ISX-mediated IL6-induced expression of the tryptophan catabolic enzymes Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase in hepatocellular carcinoma cells, resulting in an ISX-dependent increase in the tryptophan catabolite kynurenine and its receptor aryl hydrocarbon receptor (AHR). Activation of this kynurenine/AHR signaling axis acted through a positive feedback mechanism to increase ISX expression and enhance cellular proliferation and tumorigenic potential. RNAi-mediated attenuation of ISX or AHR reversed these effects. In an IDO1-dependent manner, ectopic expression of ISX induced expression of genes encoding the critical immune modulators CD86 (B7-2) and programmed death ligand-1 (PD-L1), through which ISX conferred a significant suppressive effect on the CD8

The level of global DNA methylation may be related to the cerebrovascular disease. The methylation level of Long Interspersed Nucleotide Element 1 (LINE-1) can represent the global methylation level. We investigated the association between the methylation levels of LINE-1 and ischemic stroke in Chinese. Two hundred and eighty patients of ischemic stroke and 280 age- and sex-matched controls were enrolled. The mean percentage of three CpG sites of LINE-1 was calculated for each subject and was used for analysis. Twenty four samples were re-checked for reproducibility of the methylation assay. Multivariate regression model was used to estimate the odds ratio of stroke risk for one percent change of methylation. Sex-specific analysis was also conducted. Thirty-two cases and 11 controls did not pass the methylation assay criteria, and were excluded from further analysis. The intra-class correlation has a coefficient of 0.97 for the methylation assay. The stroke cases had a lower methylation level than controls (p=0.002), especially male subjects (p=0.001). Sex-specific analysis showed that a decrease of 1% methylation level in men could increase a stroke risk by 1.2-fold after adjusting for other risk factors. LINE-1 methylation levels did not have a significant association with stroke in women. The present study shows that a lower level of LINE-1 methylation was associated with a higher risk for ischemic stroke in men, but methylation level in women did not affect the stroke risk. Our finding in Chinese is consistent with a previous result based on elderly white men.

Although morphological and anatomical studies indicate that varicose veins are characterized by venous wall weakening and subendothelial fibrosis, the exact underlying biochemical mechanism of their development remains unknown. Additionally, no quantitative proteomic study of venous proteins leading to decreased contractility of varicose veins has been reported to date. Therefore, to elucidate the molecular mechanism of altered vascular contractility, this study performed shotgun proteomic analysis to obtain protein expression profiles in patients with varicose veins. Stable isotope dimethyl labeling coupled with nanoLC-MS/MS revealed downregulation in 12 polypeptides, including myosin light chain kinase, creatine kinase B-type, ATP synthase, phosphoglycerate kinase, and pyruvate kinase. However, analyses of protein species associated with cytoskeletal assembly or with cellular morphology showed no clear up- or down-regulation. These results indicate that defects in ATP generation and utilization may account for the dysfunction of vascular smooth muscle following formation of varicose veins. Collectively, the severity of varicose veins depends on the regulatory roles of various protein factors in the metabolic coordination of physiological functions. This pilot study improves understanding of the pathogenesis of varicose veins and lays the foundation for further validation and clinical translation of biomarkers for targeted therapies in treating this disease.

Histone deacetylase 8 (HDAC8), a unique member of class I histone deacetylases, shows remarkable correlation with advanced disease stage and multiple malignant tumors However, little is known about the contribution of HDAC8 to the tumorigenesis of hepatocellular carcinoma (HCC). The present study investigated the expression of HDAC8 regulated by the aryl hydrocarbon receptor (AHR) in HCC cell lines and tissues, and the roles of HDAC8 overexpression in cell proliferation, including potentially underlying mechanisms. We assessed the correlation between the clinic-pathological parameters and the expression of AHR and HDAC8. Further, we analyzed the AHR siRNA transfection and HDAC8 inhibitors to explore the functions of HDAC8 in HCC progression in vitro and in vivo. In a panel of 289 HCC patients, HDAC8 was shown to be highly correlated with AHR expression at both mRNA and protein levels. HCC patients with high AHR expression showed a shorter survival time than that with low AHR expression. We then found that the expression of both AHR and HDAC8 was significantly upregulated in both HCC cell lines and tumor tissues compared to human normal hepatocytes and matched non-tumor tissues. Furthermore, HDAC8 inhibition remarkably inhibited hepatoma cell proliferation and transformation activity via upregulation of RB1 in vitro and in vivo. Our data revealed an important role of the AHR-HDAC8 axis in promoting HCC tumorigenesis, thus identifying HDAC8 as a potential therapeutic target for HCC treatment.

Objective Single nucleotide polymorphisms (SNPs) located at microRNA (miRNA) binding sites (miR-SNPs) can affect the expression of genes. This study aimed to identify the miR-SNPs associated with atherosclerosis and stroke. Methods Patients with ischemic stroke ( n = 657) and stroke- and myocardial infarction-free volunteers ( n = 1571) were enrolled. The carotid intima-media thickness (IMT) was measured in the control participants. Seventy-nine stroke susceptibility genes were initially selected and 13 genes were predicted to have miR-SNPs at their 3′ untranslated regions (3′UTR). The miRNA arrays were used to further identify potential miR-SNPs. The miR-SNP rs3735590 at the paraoxonase 1 (PON1) gene was finally selected and its associations with stroke and carotid IMT were evaluated. The 3′UTR reporter and SNP functional assays were then performed to validate the results. Results Compared with CC genotype, patients with CT or TT genotype at rs3735590 had lower risk of ischemic stroke (OR = 0.72, p = 0.036; OR = 0.83, p = 0.077, respectively). Among the healthy participants, the CT or TT genotype was associated with thinner IMT in the internal carotid arteries in comparison with CC genotype ( β = −0.76, p = 0.003; β = −0.022, p = 0.452, respectively). Our findings suggested that the minor allele T had a protective effect on atherosclerosis. Results from 3′UTR reporter assays showed that PON1 is a direct target gene of miR-616. In plasmid constructs carrying the risk allele C at rs3735590, miR-616 inhibited the genetic expression of PON1. However, substitution of C by T at rs3735590 reduced the miR-616 binding affinity, leading to overexpression of the PON1 gene. Conclusion Our study is the first to show that the miR-SNP at PON1 could affect genetic expression and is associated with an elevated risk for ischemic stroke and subclinical atherosclerosis.

The MMP2 gene has been implicated in the pathogenesis of endometriosis. We investigated the role and function of single-nucleotide polymorphisms (SNP) of MMP2 in relation to endometriosis. First a case-control study was conducted and 17 SNPs were examined in 211 patients and 344 controls. Regression analysis was used to evaluate the genetic effect. We used reporter assay to validate the functional consequences of the significant SNP. Two SNPs (rs243832 and rs7201) had P-values

This study computationally determines the contribution of clinicopathologic factors correlated with 5-year survival in oral squamous cell carcinoma (OSCC) patients primarily treated by surgical operation (OP) followed by other treatments. From 2004 to 2010, the program enrolled 493 OSCC patients at the Kaohsiung Medical Hospital University. The clinicopathologic records were retrospectively reviewed and compared for survival analysis. The Apriori algorithm was applied to mine the association rules between these factors and improved survival. Univariate analysis of demographic data showed that grade/differentiation, clinical tumor size, pathology tumor size, and OP grouping were associated with survival longer than 36 months. Using the Apriori algorithm, multivariate correlation analysis identified the factors that coexistently provide good survival rates with higher lift values, such as grade/differentiation = 2, clinical stage group = early, primary site = tongue, and group = OP. Without the OP, the lift values are lower. In conclusion, this hospital-based analysis suggests that early OP and other treatments starting from OP are the key to improving the survival of OSCC patients, especially for early stage tongue cancer with moderate differentiation, having a better survival (>36 months) with varied OP approaches.

Hepatitis C virus (HCV) can replicate in peripheral blood mononuclear cells (PBMCs), which can produce interferon to defend against virus infection. We hypothesized that dynamic gene expression in PBMCs might impact the treatment efficacy of peginterferon/ribavirin in HCV patients. PBMCs were collected at baseline, 1st week and 4th week of treatment from 27 chronic HCV-1 patients with 48-week peginterferon/ribavirin therapy (screening dataset n = 7; validation dataset n = 20). A sustained virologic response (SVR) was defined as undetectable HCV RNA throughout the 24 weeks after end-of-treatment. A complete early virologic response (cEVR) was defined as negative HCV RNA at treatment week 12. Forty-three differentially expressed genes identified by Affymetrix microarray were validated by quantitative polymerase chain reaction. Thirteen genes at week 1 and 24 genes at week 4 were upregulated in the SVR group compared with the non-SVR group. We selected 8 target genes (RSAD2, LOC26010, HERC5, HERC6, IFI44, SERPING1, IFITM3, and DDX60) at week 1 as the major components of the predictive model. This predictive model reliably stratified the responders and non-responders at week 1 (AUC = 0.89, p = 0.007 for SVR; AUC = 0.95, p = 0.003 for cEVR), especially among patients carrying the IL28B rs8099917 TT genotype (AUC = 0.89, p = 0.02 for SVR; AUC = 1.0, p = 0.008 for cEVR). The performance of this predictive model was superior to traditional predictors, including the rapid virologic response, viral load and IL28B genotype.

Histone deacetylase 8 (HDAC8), a unique member of class I histone deacetylases, shows remarkable correlation with advanced disease stage and multiple malignant tumors However, little is known about the contribution of HDAC8 to the tumorigenesis of hepatocellular carcinoma (HCC). The present study investigated the expression of HDAC8 regulated by the aryl hydrocarbon receptor (AHR) in HCC cell lines and tissues, and the roles of HDAC8 overexpression in cell proliferation, including potentially underlying mechanisms. We assessed the correlation between the clinic-pathological parameters and the expression of AHR and HDAC8. Further, we analyzed the AHR siRNA transfection and HDAC8 inhibitors to explore the functions of HDAC8 in HCC progression in vitro and in vivo. In a panel of 289 HCC patients, HDAC8 was shown to be highly correlated with AHR expression at both mRNA and protein levels. HCC patients with high AHR expression showed a shorter survival time than that with low AHR expression. We then found that the expression of both AHR and HDAC8 was significantly upregulated in both HCC cell lines and tumor tissues compared to human normal hepatocytes and matched non-tumor tissues. Furthermore, HDAC8 inhibition remarkably inhibited hepatoma cell proliferation and transformation activity via upregulation of RB1 in vitro and in vivo. Our data revealed an important role of the AHR-HDAC8 axis in promoting HCC tumorigenesis, thus identifying HDAC8 as a potential therapeutic target for HCC treatment.

// Shen-Nien Wang 1, 2, * , Li-Ting Wang 2, * , Ding-Ping Sun 3, 4, * , Chee-Yin Chai 5 , Edward Hsi 6 , Hsing-Tao Kuo 7, 8 , Kazunari K. Yokoyama 2, 9, 10, 11, 12, 13 , Shih-Hsien Hsu 2, 13 1 Division of Hepatobiliary Surgery, Department of Surgery, Faculty of Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 2 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Division of General Surgery, Department of Surgery, Chi-Mei Medical Center, Tainan, Taiwan 4 Department of Food Science and Technology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 5 Department of Pathology, Faculty of Medicine, College of Medicine, Kaohsiung, Taiwan 6 Department of Genome Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Internal Medicine, Division of Hepatogastroenterology, Chi-Mei Medical Center, Tainan, Taiwan 8 Department of Senior Citizen Service Management, Chia Nan University of Pharmacy & Science, Tainan, Taiwan 9 Research Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan 10 Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 11 Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 12 Faculty of Science and Engineering, Tokushima Bunri University, Sanuki, Japan 13 Center of Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan * These authors have contributed equally to this work Correspondence to: Shih-Hsien Hsu, e-mail: jackhsu@kmu.edu.tw Kazunari K. Yokoyama, e-mail: kazu@kmu.edu.tw Hsing-Tao Kuo, e-mail: kuohsu2003@yahoo.com.tw Keywords: cyclin D1 , DP1, E2F1 , hepatocellular carcinoma (HCC), ISX Received: October 05, 2015 Accepted: April 16, 2016 Published: May 09, 2016 ABSTRACT Intestine-specific homeobox ( ISX ), a newly identified proto-oncogene, is involved in cell proliferation and progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms linking gene expression and tumor formation remain unclear. In this study, we found that ISX transcriptionally activated E2F transcription factor 1 ( E2F1 ) and associated oncogenic activity by directly binding to the E2 site of its promoter. Forced expression of ISX increased the expression of and phosphorylated the serine residue at position 332 of E2F1 , which may be translocated into the nucleus to form the E2F1 –DP-1 complex, suggesting that the promotion of oncogenic activities of the ISX–E2F1 axis plays a critical role in hepatoma cells. Coexpression of ISX and E2F1 significantly promoted p53 and RB-mediated cell proliferation and anti-apoptosis, and repressed apoptosis and autophagy. In contrast, short hairpin RNAi-mediated attenuation of ISX and E2F1 decreased cell proliferation and malignant transformation, respectively, in hepatoma cells in vitro and in vivo . The mRNA expression of E2F1 and ISX in 238 paired specimens from human HCC patients, and the adjacent, normal tissues exhibited a tumor-specific expression pattern which was highly correlated with disease pathogenesis, patient survival time, progression stage, and poor prognosis. Therefore, our results indicate that E2F1 is an important downstream gene of ISX in hepatoma progression.

Lectin-like oxidized LDL receptor-1 (LOX-1) is a surface scavenger receptor for oxidized low-density lipoprotein (oxLDL). Several transcription factors have been reported to regulate LOX-1 expression. MicroRNAs are small noncoding RNAs that control gene expression, but there have been no reports of LOX-1 expression being regulated by microRNAs. Because the microRNA let-7g has been predicted to bind to LOX-1 mRNA, we investigated whether let-7g can regulate LOX-1 expression. Our experiments first demonstrated that oxLDL can reduce let-7g expression. We later confirmed that there is a let-7g binding site on the 3′-untranslated region of LOX-1 mRNA. We showed that intracellular Ca2+-activated protein kinase C is involved in the oxLDL–LOX-1–let-7g pathway. Bioinformatics predicted that the let-7g promoter has a binding site for the transcriptional repressor OCT-1. We used a promoter assay and chromatin immunoprecipitation to confirm this binding. Consequently, knockdown of OCT-1 was found to increase let-7g expression. Transfection of let-7g inhibited oxLDL-induced LOX-1 and OCT-1 expression, cell proliferation and migration. Mice fed with a high-fat diet showed a decrease in let-7g and an increase in LOX-1 and OCT-1. A study on humans showed the serum levels of let-7g are lower in subjects with hypercholesterolemia compared with normal controls. Our findings identify a negative feedback regulation between let-7g and LOX-1, and indicate that let-7g could be a target to treat cardiovascular disease.

Background Gallstone is a complex disease caused by multiple environmental and genetic factors. One of these is mucin glycoproteins. This case–control study aimed to investigate the association between single nucleotide polymorphisms (SNPs) at the MUC1-4 genes and gallstone. Methods The study included 475 cases and 941 controls. Eight tagging SNPs were selected: one at MUC1, two at MUC2, and five at MUC4. There was no available tagging SNP at MUC3. Genetic effects were initially evaluated by multivariate logistic regression. The combined effects from multiple genes were further evaluated, as well as the sex-specific effect. Permutation was used to correct for multiple testing. Results The genotypes were all in Hardy–Weinberg equilibrium. SNP rs7396030 at MUC2 yielded a p value of 0.03. Further sex-specific analysis showed significance solely with male subjects ( p = 0.005). Similarly, SNP rs4072037 at MUC1 was only significant ( p = 0.035) in males. The permutation empirical p values were 0.005 for rs7396030 and 0.02 for rs4072037. For males, the combined genetic effect yielded an OR of 4.68 ( p = 0.0008). Conclusions The SNPs at MUC1 and MUC2 are significantly associated with gallstone in men but not in women. These genes can work jointly to further increase susceptibility to gallstone in a Chinese population.

To identify candidate genes in relation to plasma lipid levels in Caribbean Hispanics.A total of 114 single nucleotide polymorphisms (SNPs) at 17 lipid-related genes were genotyped in 477 Caribbean Hispanics from the Northern Manhattan Study (NOMAS). Analyses for each SNP and haplotype were performed to evaluate the associations with four lipid traits: high- and low-density lipoprotein cholesterol (HDL-C, LDL-C), triglyceride (TG) and total cholesterol (TC).We identified 19 SNPs at 10 genes that were significantly related to lipids (p0.01), including nine involved in the reverse cholesterol transport pathway, and one involved in bile acid synthesis. Three genes, namely the apolipoprotein A5, apolipoprotein B and cytochrome p450 polypeptide 7A1 genes, accounted for the largest proportion of variation in HDL-C/TG, TC and LDL-C respectively.The cumulative effects of multiple genetic variants led to a substantially better prediction of inter-individual variations in lipid levels.

The phosphodiesterase 4D (PDE4D) gene has been reported as a risk gene for ischemic stroke. The vascular factors are between the hypothesized etiologies of sudden sensorineural hearing loss (SSNHL), and this genetic effect might be attributed for its role in SSNHL. We hypothesized that genetic variants of the PDE4D gene are associated with susceptibility to SSNHL. We conducted a case-control study with 362 SSNHL cases and 209 controls. Three single nucleotide polymorphisms (SNPs) were selected. The genotypes were determined using TaqMan technology. Hardy-Weinberg equilibrium (HWE) was tested for each SNP, and genetic effects were evaluated according to three inheritance modes. We carried out sex-specific analysis to analyze the overall data. All three SNPs were in HWE. When subjects were stratified by sex, the genetic effect was only evident in females but not in males. The TT genotype of rs702553 exhibited an adjusted odds ratio (OR) of 3.83 (95 % confidence interval = 1.46-11.18) (p = 0.006) in female SSNHL. The TT genotype of SNP rs702553 was associated with female SSNHL under the recessive model (p = 0.004, OR 3.70). In multivariate logistic regression analysis, TT genotype of rs702553 was significantly associated with female SSNHL (p = 0.0043, OR 3.70). These results suggest that PDE4D gene polymorphisms influence the susceptibility for the development of SSNHL in the southern Taiwanese female population.

The present study aims to discover single nucleotide polymorphisms (SNPs) at the apelin gene (APLN) in relation to arterial stiffness, and to explore its molecular mechanisms. A two-step genetic association study was conducted using 799 and 937 subjects in the screening and validation data, respectively. Four tagging SNPs of APLN were tested. SNP rs3115757 was significantly associated with stiffness parameters (β, Ep and PWV) in women, but not in men. The function of rs3115757 was tagged by rs3115758 which is located in miR-765 binding site in the 3′ untranslated region of APLN. The reporter assay confirmed that different alleles of rs3115758 interfered with miR-765 binding and then modified APLN expression. Over-expression of miR-765 in endothelial cells decreased mRNA and protein levels of APLN, which further inhibited the phosphorylation of eNOS and ERK/Akt/AMPK signaling. Collectively, our data showed that rs3115758 accounts for the susceptibility of arterial stiffening through miR-765-induced APLN repression.

Background.The interval between intra-arterial infusion chemotherapy (IAIC) and surgery was investigated in terms of its effects on survival in patients with locally advanced oral squamous cell carcinoma (OSCC).Methods.This retrospective study analyzed 126 patients who had completed treatment modalities for stage IV OSCC. All patients were followed up for 3 years. Kaplan-Meier and Cox regression methods were used to determine how survival was affected by general factors, primary tumor volume, TNM stage, and duration of neoadjuvant chemotherapy.Results.In 126 patients treated for locally advanced OSCC by preoperative induction IAIC using methotrexate, multivariate analysis of relevant prognostic factors showed that an IAIC duration longer than 90 days was significantly associated with poor prognosis (hazard ratio, 1.77;P=0.0259).Conclusions.Duration of IAIC is a critical factor in the effectiveness of multimodal treatment for locally advanced OSCC. Limiting the induction course to 90 days improves overall survival.

Numerous investigations underlying the hepatocellular carcinoma (HCC) diagnosis as well as detection at an early stage based on hepatitis B virus surface antigen (HbsAg) concentration in serum and aflatoxin metabolites in urine have been commonly reported in the literature. To date, these biomarkers, even though specific and accurate, are not universal for the detection of HCC elicited by all causative factors. In addition, potential biomarkers may be present at low concentrations in contrast to the presence of abundant interfering proteins with a wide dynamic range. The aim of this study is to establish an operational analysis platform of effective and noninvasive diagnostic tool with a high sensitivity to explore protein expression profiles by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) and stable isotope dimethyl labeling. Differentially expressed urinary proteins were identified and compared by the mass spectral patterns of their peptide fragments generated from protease digestion. In our results, the quantitative proteomic analysis of the differentially expressed proteins in urine identified at least 21 protein biomarker candidates with high confidence levels. We have further identified 14 proteins with up-regulation (stable isotope D/H ratio ≥ 1.5) and 7 with down-regulation (D/H ratio ≤ 0.6). The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of liver throughout progressive developments of HCC. The results would place a firm foundation for future work regarding validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various classes of HCC.