Long noncoding RNA STCAT16 suppresses cell growth and its expression predicts prognosis in patients with gastric cancer.

Gastric cancer (GC) is a leading cause of cancer­associated mortality worldwide. Previous studies demonstrated that long noncoding RNAs (lncRNAs) may be dysregulated in GC and may serve important roles in cancer progression. The present study aimed to investigate the role of the novel lncRNA stomach cancer­associated transcript 16 (STCAT16; Assembly Gene ID G038291) in the development and progression of GC. The present data suggested that the expression level of STCAT16 was decreased in GC tissues. The expression level of STCAT16 was identified to be associated with lymph node and tumour node metastasis stages. Furthermore, the expression level of STCAT16 was identified to be significantly associated with poor survival and prognosis. Knockdown of STCAT16 promoted proliferation, colony formation, migration and invasion of BGC­823 cells. In contrast, these features were suppressed in AGS cells following overexpression of STCAT16. In vivo, tumour growth was significantly decreased following STCAT16 overexpression. Collectively, the present data suggested that the lncRNA STCAT16 may act as a tumour suppressor and may inhibit GC tumour cell growth and migration. Additionally, the decreased expression level of STCAT16 was identified to be associated with poor prognosis in patients with GC.

Transplantation of mouse embryonic stem cell-derived oligodendrocytes in the murine model of globoid cell leukodystrophy.

INTRODUCTION: Globoid cell leukodystrophy (GLD) is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Cell-based therapies are highly promising strategies for GLD. In this study, G-Olig2 mouse embryonic stem cells (ESCs) were induced into oligodendrocyte progenitor cells (OPCs) and were implanted into the brains of twitcher mice, an animal model of GLD, to explore the therapeutic potential of the cells. METHODS: The G-Olig2 ESCs were induced into OPCs by using cytokines and a multi-step differentiation procedure. Oligodendrocyte markers were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The toxicity of psychosine to OPCs was determined by a cell proliferation assay kit. The GALC level of OPCs was also examined. OPCs were labeled with Dir and transplanted into the brains of twitcher mice. The transplanted cells were detected by in-Vivo Multispectral Imaging System and real-time PCR. The physiological effects of twitcher mice were assessed. RESULTS: Oligodendrocyte markers were expressed in OPCs, and 76%±5.76% of the OPCs were enhanced green fluorescent protein (eGFP)-positive, eGFP was driven by the Olig2 promoter. The effect of psychosine on cell viability indicated that OPCs were more resistant to psychosine toxicity. The GALC level of OPCs was 10.0±1.23 nmol/hour per mg protein, which was significantly higher than other cells. Dir-labeled OPCs were injected into the forebrain of post-natal day 10 twitcher mice. The transplanted OPCs were myelin basic protein (MBP)-positive and remained along the injection tract as observed by fluorescent microscopy. The level of the Dir fluorescent signal and eGFP mRNA significantly decreased at days 10 and 20 after injection, as indicated by in-Vivo Multispectral Imaging System and real-time PCR. Because of poor cell survival and limited migration ability, there was no significant improvement in brain GALC activity, MBP level, life span, body weight, and behavioral deficits of twitcher mice. CONCLUSIONS: ESC-derived OPC transplantation was not sufficient to reverse the clinical course of GLD in twitcher mice.

Differentiation of nonhuman primate embryonic stem cells into hepatocyte-like cells.

OBJECTIVE: To investigate whether cells derived from rhesus monkey embryonic stem cells (ESC) had hepatocyte characteristics after the differentiation. METHODS: Rhesus monkey ESC were induced towards hepatocyte-like cells via a four-step differentiation process: the formation of embryoid bodies (EB), EB in activin A and insulin-transferrin-selenium medium for 4 days, in fibroblast growth factor (FGF)-4 and bone morphogenetic protein-2 (BMP2) medium for 8 days, in hepatocyte culture medium containing hepatocyte growth factor for 3 days and then with oncostatin M and dexamethasone for another 5 days. Expression of albumin (ALB), glucose-6-phosphatase, α-fetoprotein (AFP) and α-1 antitrypsin (α1-AT) at the mRNA level in differentiated cells were detected by reverse transcription-polymerase chain reaction. The expression of hepatocyte markers AFP, ALB, hepatocyte nuclear factor 4 (HNF4), cytokeratin 8 (CK8), CK19 and cell proliferation marker, Ki67, in the differentiated cells were determined by immunocytochemistry. The ultrastructure of the differentiated cells was examined by electron microscopy. Indocyanine green (ICG) uptake was also explored. RESULTS: After induction, some differentiated cells were binucleate, which is typical of hepatocytes. Hepatocyte-specific genes ALB, glucose-6-phosphatase, AFP and α1-AT were expressed in the differentiated cells. The differentiated cells expressed hepatocyte markers AFP, ALB, HNF4, CK8 and CK19 at the protein level. The cells also expressed cell proliferation marker Ki67. Under electron microscopy, the ultrastructures of hepatocyte-like cells, such as mitochondrion and catalase-containing peroxisomes, were observed in the differentiated cells. ICG uptake test was positive in differentiated cells. CONCLUSIONS: With cytokine induction, rhesus monkey ESC differentiated into cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes.

Significance of viral status on recurrence of hepatitis B-related hepatocellular carcinoma after curative therapy: A meta-analysis.

AIM: The impact of viral status on recurrence of hepatitis B-related hepatocellular carcinoma (HCC) after curative therapy remains controversial. This meta-analysis aimed to determine whether the presence of viral load, genotype, specific mutation and antiviral therapy influenced HCC recurrence after curative therapy. METHODS: We performed a meta-analysis including 20 studies to assess the effect of viral status and antiviral therapy with nucleoside analog on recurrence of HCC after curative therapy. The pooled odds ratios (OR) were calculated using a random or fixed effects model. PUBMED, MEDLINE, EMBASE and the Cochrane Database were searched for articles published from 1990 to December 2012. RESULTS: Our results showed that the presence of high viral load significantly increased overall HCC recurrence risk after curative therapy. Pooled data from four studies on the recurrence rate among patients with genotype C infection compared with genotype B showed an increased risk of recurrence. Basal core promoter (BCP) mutation was associated with a significant risk in the recurrence of HCC. The pooled estimate of treatment effect was significantly in favor of a preventive effectiveness of antiviral therapy. CONCLUSION: The present study suggested that HCC patients with high viral load, genotype C and BCP mutation had a significantly higher risk of recurrence. Antiviral therapy has potential beneficial effects after the curative treatment of HCC in terms of tumor recurrence.

Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza-2'-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase-independent apoptosis in gastric cancer cells.

The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, -8 and -9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.

Effects of interferon therapy on development of hepatocellular carcinoma in patients with hepatitis C-related cirrhosis: A meta-analysis of randomized controlled trials.

AIM: The role of interferon (IFN) therapy on prevention of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related cirrhosis remains controversial. This meta-analysis aimed to determine whether IFN therapy reduced the incidence of HCC in HCV-related cirrhotic patients. METHODS: We performed a meta-analysis including eight randomized controlled trials (RCT) (a total of 1505 patients) to assess the effect of IFN therapy on prevention of HCC in patients with HCV-related cirrhosis. The pooled odds ratios (OR) were calculated using a random or fixed effects model. RESULTS: Our results showed that IFN therapy significantly decreased the overall HCC incidence in HCV-related cirrhotic patients (OR, 0.29; 95% confidence interval [CI], 0.10-0.80; P = 0.02). HCC risk in patients who failed to achieve sustained virological response (SVR) in the initial IFN-based treatment was also reduced by maintenance IFN therapy (OR, 0.54; 95% CI, 0.32-0.90; P = 0.02). Subgroup analysis indicated that IFN therapy decreased HCC incidence in HCV-related cirrhotic patients during long-term follow up (>48 months) evidently (OR, 0.25; 95% CI, 0.09-0.67; P = 0.006). However, subgroup analysis of four RCT with short-term follow up (≤48 months) did not demonstrate the significant difference in HCC incidence between IFN-treated cirrhotic patients and controls (OR, 0.78; 95% CI, 0.39-1.55; P = 0.48). CONCLUSION: The present study suggested that IFN therapy could efficiently reduce HCC development in patients with HCV-related cirrhosis; this effect was more evident in the subgroup of patients with long-term follow up (>48 months). Patients who received maintenance IFN therapy had a lower risk of HCC than controls.

Differentiation of nonhuman primate embryonic stem cells along neural lineages.

The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14-16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), beta-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and beta-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, beta-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen beta-tublinIII (73+/-4.7%) and some expressed intermediate neuron antigen MAP2 (18+/-7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%+/-1.2%), GFAP (4.93+/-1.9%), S100 (7+/-3.5%) and O4 (0.27+/-0.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.

Treatment of surgically induced acute liver failure by transplantation of HNF4-overexpressing embryonic stem cells.

OBJECTIVE: Tissue-specific stem cells from differentiating embryonic stem (ES) cells are both pluripotent and genetically flexible. Recent observations indicate that ES cells can differentiate into hepatocytes. Therefore, cell-based therapy can potentially be a therapeutic alternative to liver transplantation. In this study the treatment of acute liver failure in rats by transplantation of hepatocyte nuclear factor 4 (HNF4)-overexpressing ES cells was investigated. METHODS: The HNF4 was transfected into ES cells and ES cell clones overexpressing HNF4 were selected. The levels of markers of hepatocyte differentiation, including albumin, transthyretin, glucose-6-phosphates (G-6-P) and SAPK/ERK kinase-1 (SEK1) mRNA, were tested in spontaneously differentiated HNF4-overexpressing ES cells by reverse transcription-polymerase chain reaction (RT-PCR). The ultrastructure of the spontaneously differentiated HNF4-overexpressing ES cells was examined by electron microscopy. To induce acute liver failure, Sprague-Dawley rats were subjected to 90% hepatectomy and given 5% oral dextrose. The rats were divided into three groups. The rats in the treatment group (n = 12) received intraliver injection of 2 x 10(7) undifferentiated HNF4-overexpressing ES cells from the same clone, the rats in control group 1 (n = 12) received 2 x 10(7) undifferentiated ES cells, and the rats in control group 2 (n = 12) received the same volume of media without any cells. RESULTS: All rats in control group 1 and control group 2 died within 72 h, while 33% of rats that received undifferentiated HNF4-overexpressing ES cells transplantation survived more than 1 month. Spontaneously differentiated HNF4-overexpressing ES cells only expressed transthyretin mRNA. The cells were rich in mitochondrion and catalase-containing peroxisomes in ultrastructure. CONCLUSIONS: Transplantation of ES cells could be a potential treatment in supporting life during acute liver insufficiency and could be a bridge to orthotopic liver transplantation.

[Clinical significance of serum GPDA-F determined by immunoelectrophoresis in diagnosis of hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Serum fast band of glycylproline dipeptidyl aminopeptidase isoenzyme (GPDA-F) is useful to the diagnosis of hepatocellular carcinoma, especially for the cases without expression of alpha-fetoprotein (AFP). Polyacrylamide electrophoresis for detection of GPDA-F is relatively complicated and has limitation in its clinical use. This study was to establish a simple and easy method of immunoelectrophoresis to detect serum GPDA-F, and evaluate clinical value of GPDA-F in the diagnosis of hepatocelluar carcinoma. METHODS: Serum GPDA-F was purified to raise polyclonal GPDA-F antibody, and immunoelectrophoresis was established for the detection of serum GPDA-F. Serum GPDA-F in 99 specimens of hepatocellular carcinoma and 115 specimens of benign liver diseases (36 cases of liver cirrhosis, 23 cases of acute hepatitis, 38 cases of chronic hepatitis, and 18 cases of benign liver space-occupying lesions) was simultaneously detected by both polyacrylamide electrophoresis and immunoelectrophoresis. The clinical value of serum GPDA-F detected by immunoelectrophoresis was compared with that by polyacrylamide electrophoresis for the diagnosis of hepatocellular carcinoma. RESULTS: If the cut-off was set at 71 u/L, the diagnostic sensitivity, specificity, and accuracy of immunoelectrophoresis in detecting GPDA-F in hepatocellular carcinoma were 83.8%, 85.2%, and 84.6%, respectively; while those of polyacrylamide electrophoresis were 81.8%, 77.3%, and 79.4%, respectively. CONCLUSIONS: Serum GPDA-F detected by immunoelectrophoresis is useful to the diagnosis of hepatocellular carcinoma. Compared with polyacrylamide electrophoresis, this method is cheap, time-saving, and easy.

Generation of hepatocytes from cultured mouse embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and beta-nerve growth factor (beta-NGF). RA, HGF, and beta-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of alpha1-antitrypsin (alpha1-AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and beta-NGF to the cell culture, many epithelioid cells were noticed. alpha1-AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and beta-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited.