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.

[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.

[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.

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs.

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would 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.