Decreased expression of KLF6 and its significance in gastric carcinoma.

To study the expression of the Krüppel-like transcription factor 6 (KLF6) in human gastric carcinoma and normal gastric mucosa tissues, and to explore the role of KLF6 in the carcinogenesis and tumor progression and its clinical significance. Expression of KLF6, P21WAF1 and PCNA was investigated by immunohistochemistry for 69 surgically resected gastric carcinoma tissues and corresponding normal gastric mucosa tissues, respectively. The correlations of KLF6 expression with clinicopathological characteristics, P21WAF1 and PCNA were examined. Positive-expression of KLF6 was 64 out of 69 cases (92.8%) in normal gastric mucosa and only 23 cases (33.3%) in gastric carcinoma. Expression of KLF6 in the gastric carcinoma was remarkably lower than normal gastric mucosa. Decreased expression of KLF6 in gastric carcinoma was significantly associated with histological differentiation (P<0.01), TNM stage (P<0.05), lymph node metastasis (P<0.01) and distant metastasis (P<0.05). There was no significant correlation between KLF6 expression and sex, age. Meanwhile, expression of KLF6 was associated with expression of P21WAF1 in both normal gastric mucosa and gastric carcinoma (P<0.05). In addition, decreased expression of KLF6 in gastric carcinoma was positively associated with PCNA level (r=0.719, P<0.01) by association analysis. Down-regulation of KLF6 might play an important role in the carcinogenesis and development of human gastric carcinoma and have significant clinical value.

Endoscopic diagnosis and treatment of biliary leak in patients following liver transplantation: a prospective clinical study.

BACKGROUND: Orthotopic liver transplantation has been widely used in patients with end-stage liver disease within the last two decades. However, the prevalence of biliary complications after liver transplantation remains high. The most common short-term biliary complication may be biliary leak. So, we examined 13 patients with biliary leak after liver transplantation, attempting to evaluate the role of endoscopic diagnosis and treatment of biliary leak and the incidence of bile duct stricture after healing of the leak. METHODS: Six cases of T-tube leak and seven cases of anastomosis leak complicating liver transplantation were enrolled in this prospective study. Six patients were treated by endoscopic plastic stent placement, two by nasobiliary catheter drainage, two by papillosphincterotomy, and three by nasobiliary catheter drainage combined with plastic stent placement. Some patients received growth hormone treatment. RESULTS: The bile leak resolution time was 10-35 days in 10 patients with complete documentation. The median time of leak resolution was 15.3 days. Four cases of anastomosis stricture, three cases of common hepatic duct and one case of multiple bile duct stenosis were detected by follow-up nasobiliary catheter cholangiography or endoscopic retrograde cholangiopancreatography. CONCLUSIONS: Endoscopic nasobiliary catheter or plastic stent placement is a safe and effective treatment for bile duct stricture occurring after bile leak resolution in most liver transplantation patients. Nasobiliary catheter combined with plastic stent placement may be the best choice for treating bile leak, because, theoretically, it may prevent the serious condition resulting from accidental nasobiliary catheter dislocation, and it may have prophylactic effects on upcoming bile duct stricture, although this should be further confirmed.

Effects of YC-1 on hypoxia-inducible factor 1-driven transcription activity, cell proliferative vitality, and apoptosis in hypoxic human pancreatic cancer cells.

OBJECTIVES: To investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on HIF-1-driven transcription activity, cell proliferative vitality, and apoptosis in hypoxic human pancreatic cancer cells. METHODS: Human pancreatic cancer PC-3 cells were incubated under normoxic or hypoxic conditions. YC-1 was added to the media with different concentrations. The HIF-1alpha protein expression was detected by means of immunocytochemical staining and Western blotting. Semiquantitative reverse transcriptase polymerase chain reaction was used to determine the mRNA expression of HIF-1alpha, vascular endothelial growth factor (VEGF), and glucose phosphate isomerase (GPI). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were used to detect the cells' proliferative vitality and apoptosis. RESULTS: Hypoxic PC-3 cells expressed a higher level of HIF-alpha protein in nucleus compared with the normoxic controls. When the dose of YC-1 was at 100 micromol/L, the expression location of HIF-alpha shifted from nucleus to cytoplasm. Western blotting revealed that YC-1 reduced the level of HIF-1alpha protein expression, and the inhibitory effect was dose dependent. Moreover, YC-1 dose dependently inhibited mRNA expression levels of VEGF and GPI in hypoxic cells. YC-1 inhibited proliferative vitality and induced apoptosis of hypoxic PC-3 cells in a dose-dependent manner. CONCLUSIONS: YC-1 inhibits HIF-1alpha expression in hypoxic pancreatic cancer cells, which is accompanied by the translocation of HIF-1alpha from nucleus to cytoplasm, decreased mRNA expression of VEGF and GPI, reduced cell proliferative vitality, and increased apoptosis. These results suggest that HIF-1 is a potential therapeutic target for pancreatic cancer.

[Inhibitory effect of YC-1 on induction of VEGF and GPI genes in hypoxic human pancreatic cancer cells].

OBJECTIVE: To investigate the function and mechanism of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on activity of VEGF and GPI genes in human pancreatic cancer PC-3 cells incubated under hypoxic conditions. METHODS: Human pancreatic cancer PC-3 cells were incubated under hypoxic culture conditions. Immunocytochemical staining was used to detect HIF-1alpha protein expression in hypoxic and normoxic PC-3 cells. Semi-quantitative RT-PCR was used to detect the effect of YC-1 on the expression of VEGF and GPI mRNA and HIF-1alpha protein in PC-3 cells. Effect of YC-1 on the expression of HIF-1alpha protein was examined by Western blotting. MTF assay was used to detect proliferation of hypoxicPC-3 cells. RESULTS: HIF-1alpha expression was mainly located in nuclei in hypoxic PC-3 cells. The mRNA synthesis of VEGF and GPI and the protein expression of HIF-1alpha were significantly decreased in the group treated with the highest concentration of YC-1 (100 micromol/L). Compared to placebo, YC-1 inhibited the proliferation of hypoxic PC-3 cells greatly when it was increased to 100 micromol/L. CONCLUSION: YC-1 inhibited the transcription of VEGF and GPI in hypoxic human pancreatic cancer PC-3 cells. It was induced by down-regulation of HIF-1alpha protein. YC-1 inhibites the proliferation of PC-3 cells exposed to hypoxic conditions.

Retrospective survey of 452 patients with inflammatory bowel disease in Wuhan city, central China.

BACKGROUND: Inflammatory bowel disease (IBD) had been uncommon in China until about 1990, but since then, it has been seen in the clinical setting more and more. The prevalence and phenotype of IBD in the Chinese population is not well known. The present study investigates the trend of prevalence in ulcerative colitis (UC) and Crohn's disease (CD) in Wuhan City, central China, and evaluates clinical features, extraintestinal manifestations, and the treatment of IBD in the last 14 years. METHODS: Three hundred and eighty-nine patients with UC and 63 patients with CD were retrospectively collected from 5 central hospitals in Wuhan City, in which high-quality endoscopic and histological diagnoses were available from 1990 to 2003. UC and CD were diagnosed based on clinical, experimental, radiological, endoscopic, and histological examinations according to the internationally accepted Lennard-Jones criteria. RESULTS: The trend toward prevalence of UC and CD increased between 1990 and 2003 in Wuhan City. There was no change in the sex and age distribution comparing 1990 to 1996 with 1997 to 2003 both in UC and CD. However, the number of individuals with higher education and a professional occupation during 1997 to 2003 was significantly higher than that during the period 1990 to 1996 in patients with UC (OR 2.1, 95% CI 1.27-3.35, P = 0.004; OR 2.2, 95% CI 1.31-3.61, P = 0.003). The mean age of patients with CD was significantly younger than that of UC at the time of diagnosis (32.6 +/- 12.5 vs. 42 +/- 14.5, P < 0.0001). The ratio of male to female patients was 1.53:1 in UC and 2.32:1 in CD, respectively. The mean duration of onset of the disease to diagnosis was 1.4 years in UC and 1.1 years in CD. The extra intestinal manifestations of UC and CD were 5.7% and 19%, respectively, and complications of UC and CD were 6.4% and 50.8%, respectively. Only 3% of UC patients required surgery, whereas 27% of CD patients underwent surgical procedures (P < 0.001). CONCLUSION: The prevalence of IBD has increased in Wuhan City, central China, but is not as high as in Western countries. The disease in Wuhan City has often been associated with young adult professional males with a high level of education. The clinical presentation of UC was often mild and had few extra intestinal manifestations.

Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 in cell-free system and in hepatic stellate cells.

Transforming growth factorbeta1 (TGFbeta1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFbeta1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 were designed to down-regulate TGFbeta1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFbeta1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFbeta1 in many cellular processes and a potential therapeutic candidate for TGFbeta1-related diseases.

[Hammerhead ribozyme-mediated cleavage of transforming growth factor beta1 RNA in a cell-free system and in hepatic stellate cells].

OBJECTIVE: To identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs). METHODS: The ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression. RESULTS: The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression. CONCLUSION: The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.

Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats.

BACKGROUND/AIMS: Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats. METHODS: Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride. RESULTS: TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride. CONCLUSIONS: The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.

AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro. METHODS: Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis. RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either. CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

Preparation and identification of anti-transforming growth factor beta1 U1 small nuclear RNA chimeric ribozyme in vitro.

AIM: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro. METHODS: TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE. RESULTS: Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct. CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.

AIM: To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro. METHODS: HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. 32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T7 RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with 32p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis. RESULTS: HpRz has the specific ability of cleavage of target RNA at 37 degrees and 12 mM MgCl2. Km=26.31 nmol/L, Kcat=0.18/min. These results revealed that the design of HpRz was correct. CONCLUSION: HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.

[Tumor necrosis factor alpha HLA-DRB(1) gene polymorphism and genetic susceptibility to cirrhosis].

OBJECTIVE: To study the relationship between the gene polymorphism of HLA-DRB(1) and tumor necrosis factor (TNF)alpha with the genetic susceptibility to cirrhosis. METHODS: The gene polymorphism of DRB(1) and TNF alpha of 106 cases of cirrhosis due to HBV and 108 controls were detected by means of polymerase chain reaction-sequence specific primer and RFLP techniques. RESULTS: The frequency of DRB(1) * 120X and TNF2/1 was increased in the patients as compared with the controls (35.9% vs 11.1%, P < 0.001, 19.8% vs 10.2%, P < 0.05 respectively), The frequency of DRB(1) * 150X allele was reduced in the patients as compared with the controls (13.2% vs 30.6%, P < 0.05). It is suggested that the gene polymorphism of HLA-DRB(1) and TNF alpha to be associated with genetic susceptibility to cirrhosis. Cross analysis showed that DRB(1) * 120X allele was more strongly associated with genetic susceptibility to cirrhosis than TNF2 allele. CONCLUSIONS: The genetic susceptibility to cirrhosis is associated with DRB(1) * 120X and TNF2 allele, persons with DRB(1) * 120X and TNF2 allele have an increased risk for the liver cirrhosis occurrence; DRB(1) * 120X allele may be a susceptibility gene to liver cirrhosis and DRB(1) * 150X allele may be a protective gene from liver cirrhosis.

The endoscopic retrograde cholangiopancreatographic manifestations of histopathologically diagnosed hepatocellular carcinoma with obstructive jaundice.

To study the manifestations of endoscopic retrograde cholangiopancreatography (ERCP) in patients of obstructive jaundice associated with HCC, 32 cases of histopathologically diagnosed HCC with obstructive jaundice were successfully examined with routine ERCP. 31 patients were demonstrated by ERCP as having malignant obstructive jaundice. Among them, 19 were hepatic perihilar bile duct stricture, 7 bile ductile tumorous thrombus, 3 perihilar bile duct stricture complicated with thrombus, 2 metastasis to hilar lymph node, and 1 common bile duct stone as proven by sphincterotomy. The malignant perihilar stricture was all of type III and IV by Bismuth standard of Klastin tumor. In patients identified as having bile duct tumor thrombus, by the Ueda classification, none was of type I and II; 1 type III a; 4 III b; 2 type IV. HCC with obstructive jaundice was mainly caused by the malignant infiltration of tumor, and most stricture was of serious nature. When major extra-hepatic bile duct was involved by tumor thrombus, obstructive jaundice might develop. Malignant perihilar stricture and tumor thrombus might coexist in some patients. Jaundice was rarely caused by hepatic hilar lymph node metastasis. Jaundice was not necessarily caused by tumors and sometimes, it might be caused by common bile stones. Care should be exercised in differentiation diagnosis in such patients.