Background and purpose:Urothelial carcinoma of the bladder (UCB) is the most common cancer in urinary system. Yes associated protein (YAP) gene is closely associated with urothelial carcinoma of the bladder. The study was aimed to explore the effect of siRNA targeting the YAP gene on cell proliferation and migration of T24 cells. Methods:Small interfering RNA (siRNA) was transfected together with LipofectamineTM2000 in T24 human bladder cancer cells to block the YAP signal pathway. The effect of siRNA on cell proliferation and invasiveness was assessed by cell counting kit-8 (CCK-8) assay, Transwell migration assay and wound healing assay. Quantitative real time-Polymerase chain reaction (qRT-PCR) and Western blot analysis were used to conifrm the successful suppression of YAP gene and protein by siRNA. Results:Expression of YAP gene and protein was successfully suppressed after transfected with siRNA which verified by qRT-PCR and Western blot(RNA:F=93.91, P<0.000 1; Protein: F=4.62, P<0.05). As CCK-8 test showed, the proliferation of T24 bladder cancer cells was successfully restrained by inhibition of YAP gene compared with blank control and negative control(12 h: F=6.00, P=0.037;24 h: F=41.72, P=0.000 3;36 h:F=462.8, P<0.000 1;48 h:F=236.6, P<0.000 1;72 h:F=140.5, P<0.000 1). Transwell and wound healing test were performed after YAP gene was interfered by siRNA. The result demonstrated that migration of T24 bladder cancer cells was signiifcantly inhibited (Transwell: F=43.55, P<0.05;Wound healing: F=43.55, P<0.05). Conclusion:This study suggested that YAP gene was an important enhancer for the proliferation and migration of bladder cancer cells.

It was constructed that a genomic DNA library from Lactobacillus sp. MD-1 yielding D, L-lactic acid. The gene encoding L-lactate dehydrogenase (L-LDH) was cloned from the genomic library of strain MD-1 by complementation in E. coli FMJ144 which was lactate dehydrogenase and pyruvate-formate lyase double defective mutant. The nucleotide sequence of the ldhL gene predicted a protein of 316 amino acid starting with ATG. The putative molecular weight of the L-LDH amino acid sequence was 33.84kD. A putative typical promoter (-35 and -10 boxes) had been observed in the 5' noncoding region. An rho-independent transcriptional terminator has been observed in the 3' noncoding region. Three highly conserved regions (Gly13 approximately Asp50, Asp73 approximately Ileul00 and Asn123 approximately Arg154) with several conserved residues had been identified. Gly13 approximately Asp50 was NADH-binding site domain. Asp73 approximately Ileu100 and Asn123 approximately Arg154 were reported to be the active site domains. The ldhL and the L-LDH of Lactobacillus sp. MD-1 showed the low identity and similarity with other Lactobacilli, and the highest percentage were 61.9% and 68.9% respectively. All the above indicated this gene is a novel ldhL.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; L-Lactate Dehydrogenase ; chemistry ; genetics ; physiology ; Lactobacillus ; genetics ; Molecular Sequence Data

ABSTRACT:OBJECTIVE To investigate the role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx). METHODS We analyze the expression of Cx26, Cx30, Cx32 and Cx43 in human specimens consisting of: Normal cervix (n=78), CaCx FIGO stage Ⅰ (n=148), CaCx FIGO stage Ⅱ (n=165). InCaCx cell lines, Hela- Cx32 (induced expression by doxycycline), C- 33A (endogenously express Cx32) and siHa (transiently transfected plasmid with Cx32), we detected the role of Cx32 against tostreptonigrin/cisplatin-induced apopotosisin presence or absence of functional GJ through using GJ inhibitors or low density cultural.Furtherly, we observed the relativity of Cx32 and EGFR expression in human specimens. Also, we detected the role of EGFR signaling pathway in the process of Cx32 anti-apoptosis through suppressed EGFR expression by inhibitors or siRNA sequences in cell lines. RESULTS We firstly demonstrated the expression of Cx32 was highly upregulated and accumulated in cytoplasm in the CaCx specimens, and the degree of upregulation correlated with advanced FIGO stages. Thus,in three human cervical cell lines, Cx32 was shown to suppress apoptosis when GJ formation is inhibited. No matter in cases of CaCx or cell lines, Cx32 expression was highly correlated with expression of EGFR and the EGFR pathway is an essential component of the Cx32-induced anti-apoptotic effect. CONCLUSION Cx32, traditionally tumor suppressive protein, was shown to be tumor protective against chemotherapy through EGFR pathway in a GJ-independent way.

OBJECTIVETo observe the effect of octreotide (OCT) on inhibiting hepatocellular carcinoma (HCC) and investigate its mechanisms.

METHODSNude mice bearing xenografts in situ were treated with OCT or saline control for 7 weeks since tumor implantation. The immunohistochemistry for somatostatin receptor 2 (SSTR2), cMet, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, Smad4 and Smad7 was performed. SSTR2 and Smad4 mRNA expression was measured by semi-quantitative RT-PCR.

RESULTSAfter OCT treatment, the mean tumor weight in mice given OCT (0.17 +/- 0.14 g) was significantly lower than that of the control group (0.53 +/- 0.06 g). The inhibition rate of tumor was 67.9%. mRNA and protein expression of SSTR2, Smad4 in tumor cells of the treatment group were significantly more than that of the control group. cMet expression in OCT group was remarkably lower than that in control group. Between two groups, the expression of TGFbeta1, phospho-Smad2 and Smad7 were not remarkably different. In addition, phospho-Smad2 expression in HCC was significantly less than that of the normal hepatic cell.

CONCLUSIONOCT can inhibit the growth of HCC xenografts markedly. The mechanisms of OCT-induced inhibition effect may be related to up-regulating SSTR2 expression, down-regulating cMet, and recovering the function of TGFbeta/Smads-induced antitumor. In addition, the decreased expression of phospho-Smad2 may be an important feature of Bel7402 cells.

Animals ; Antineoplastic Agents, Hormonal ; therapeutic use ; Humans ; Liver Neoplasms, Experimental ; drug therapy ; metabolism ; pathology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Octreotide ; therapeutic use ; Proto-Oncogene Proteins c-met ; biosynthesis ; Receptors, Somatostatin ; biosynthesis ; Smad2 Protein ; biosynthesis ; Transforming Growth Factor beta ; biosynthesis

OBJECTIVETo investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis.

METHODSFresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR.

RESULTSCirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue.

CONCLUSIONHepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.

Animals ; Diethylnitrosamine ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo investigate the effects of two histone deacetylase (HDAC) inhibitors, valproic acid (VPA) and TSA, on the expression of vascular endothelial growth factor (VEGF) and its receptor KDR of the leukemia cell line Kasumi-1 cells, and to explore their potential mechanism in leukemia angiogenesis.

METHODKasumi-1 cells were treated with VPA and TSA at different concentrations for 3 days. The mRNA and protein expression levels of VEGF and KDR were determined by semi-quantitative RT-PCR and Western blot, and the bFGF mRNA by semi-quantitative RT-PCR.

RESULTSAs compared with that of control groups, VPA at 3 mmol/L downregulated the VEGF mRNA expression level for VEGF(121) from 0.632 ± 0.014 to 0.034 ± 0.004 and for VEGF(165) from 0.526 ± 0.021 to 0.015 ± 0.001, for KDR mRNA from 0.258 ± 0.034 to 0.038 ± 0.000, and for bFGF mRNA from 0.228 ± 0.017 to 0.086 ± 0.015. TSA downregulated the VEGF mRNA and KDR mRNA at concentration of 100 nmol/L, but its effect on bFGF mRNA only at higher concentration.

CONCLUSIONHDAC inhibitors might inhibit the leukemia angiogenesis by regulating the expression of VEGF and its recptor.

Angiogenesis Inducing Agents ; Cell Line ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate the pathogenesis of ischemic-type biliary lesions (ITBLs) in post-liver transplant patients and the possible therapeutic mechanisms of sirolimus.

METHODSThe clinic data of 32 post-liver transplant patients with ITBLs from May 2004 to December 2010 was analyzed. There were including 25 male and 7 female patients with a median age of 46 years (ranging from 19 to 61 years). Patients were divided into those who received sirolimus (sirolimus group) and those who did not (control group). The expression of IL-2, FoxP3, and IL-10 in the portal area, liver function indexes, and bile duct injury score were assessed pre-ITBL, when ITBLs were identified, and after 6 months of sirolimus treatment.

RESULTSCompared with pre-ITBL optical density (OD) values, there was a significantly increase in IL-2 OD(0.138 ± 0.050 in control group and 0.141 ± 0.052 in sirolimus group), but not FoxP3 and IL-10 OD in both groups at the time ITBLs were diagnosed. After 6 months of treatment, the IL-2, FoxP3, and IL-10 OD values in the control group were not different from those when ITBLs were diagnosed. There was a significant reduction in post-therapy IL-2 OD(0.107 ± 0.043, t = 2.087, P = 0.044), and a significant elevation in FoxP3(0.213 ± 0.039) and IL-10 OD(0.187 ± 0.048) in sirolimus group as compared with those when ITBLs were diagnosed(t = -3.822 and -4.350, both P < 0.01). There was a significant increase in serum levels of ALT, AST, total bilirubin, γ-glutamyl transpeptidase and ALP at the time ITBLs were diagnosed compared with pre-ITBL levels in both groups. After 6 months of treatment, the above indexes had not changed in the control group, but significantly improved in the sirolimus group, and the bile duct injury score in the sirolimus group had significantly decreased(4.4 ± 2.4, Z = -2.568, P = 0.010). The 1-year and 3-year graft survival rates in the control group were 6/13 and 5/13, respectively, and 17/19 and 13/19, respectively, in the sirolimus group (χ(2) = 7.166, P = 0.007; χ(2) = 5.398, P = 0.020, respectively).

CONCLUSIONSSirolimus can downregulate IL-2 expression and upregulate FoxP3 and IL-10 expression, thereby stimulating FoxP3+ Treg cells, suppressing immunopathological damage, and promoting epithelial repair in bile ducts.

Adult ; Bile Duct Diseases ; drug therapy ; Female ; Forkhead Transcription Factors ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Ischemia ; diet therapy ; Liver Transplantation ; Male ; Middle Aged ; Postoperative Complications ; drug therapy ; Sirolimus ; therapeutic use ; Young Adult

OBJECTIVETo investigate the characteristics of diagnosis and treatment of iatrogenic ejaculatory duct obstruction following prostatic hyperthermia.

METHODSRetrospective analyses were made of the clinical data of 3 cases of ejaculatory duct obstruction following prostatic hyperthermia.

RESULTSThe 3 cases were diagnosed as urethrostenosis and deformity of the posterior urethra by transurethral ultrasound and semen analysis. And all the 3 patients had undergone prostatic hyperthermia for prostatitis. Transurethral resection of the ejaculatory duct (TURED) was performed to remove the obstruction and the postoperative semen analysis showed both semen volume and sperm count to be normal.

CONCLUSIONUrethra microwave thermotherapy, urethra radiofrequency, or per urethra rheophore ablation can be adopted in the treatment of protatitis, but should be strictly indicated and cautiously selected lest secondary iatrogenic ejaculatory duct obstruction should result. For the treatment of this obstruction, TURED is the first choice.

Adult ; Constriction, Pathologic ; etiology ; surgery ; Ejaculatory Ducts ; surgery ; Humans ; Hyperthermia, Induced ; adverse effects ; Iatrogenic Disease ; Male ; Prostatitis ; therapy ; Retrospective Studies

OBJECTIVETo investigate in vivo inhibitory effect of histone deacetylase (HDAC) inhibitor valproic acid (VPA) on xenografted Kasumi-1 tumor in nude mice and its mechanism.

METHODSXenografted Kasumi-1 tumor mouse model was established by subcutaneous inoculation of Kasumi-1 cells. Xenotransplanted nude mice were assigned into control or VPA treatment groups. Volume of the xenografted tumors was measured and compared between the two groups. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was applied to detection of tumor cell apoptosis. The gene expression of GM-CSF, HDAC1, Ac-H3 and survivin was studied with semi-quantitative RT-PCR and Western blotting. ChIP method was used to assay the effects of VPA on acetylation of histone H3 within GM-CSF promoter region.

RESULTS(1) VAP significantly inhibited xenografted Kasumi-1 tumor growth. The calculated inhibition rate was 57.25%. (2) Morphologic study showed that VPA induced differentiation and apoptosis of Kasumi-1 tumor cells. The apoptosis index of VAP treatment group [(3.661 +/- 0.768)%] was significantly higher than that of control group [(0.267 +/- 0.110)%]. (3) Comparing to those in control group, the level of nuclear HDAC1 protein was significantly decreased, the Ac-H3 protein expression level was increased, the mRNA and protein expression levels of GM-CSF and acetylation of histone H3 were remarkably increased, and the gene expression level of survivin significantly decreased in VPA treatment group.

CONCLUSIONVAP significantly inhibits xenografted Kasumi-1 tumor growth and induces tumor cell differentiation and apoptosis. The mechanism may be decrease of survivin gene expression, inhibition of nuclear expression of HDAC, promotion of histone protein acetylation level and acetylation of histone H3 within GM-CSF promoter region, and increase of GM-CSF transcription.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Mice ; Mice, Nude ; Valproic Acid ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo observe the impact of Roux-en-Y gastric bypass on quality of life in non-obese patients with type 2 diabetes mellitus.

METHODSThirty-seven non-obese patients with type 2 diabetes mellitus who underwent Roux-en-Y gastric bypass were prospectively studied. A 36-item short form healthy survey questionnaire(SF36), the diabetes treatment satisfaction questionnaire(DTSQ), and quality of life scale for patients with type 2 diabetes mellitus(DMQLS) were used to evaluate the quality of life for all the non-obese patients with type 2 diabetes mellitus preoperatively and 12 months postoperatively.

RESULTThe blood glucose and lipid indexes were significantly decreased after operation(all P<0.05). SF36 showed the physical and mental synthesis scores at 12 month after operation were 74.6±18.3 and 79.8±14.9 respectively, higher than those at one week before operation(54.9±15.1 and 56.4±17.8, both P<0.01). DTSQ showed treatment satisfaction score was increaced significantly after operation(29.2±7.1 vs. 15.4±5.6, P<0.01). The quality of life evaluated by DMQLS, was also significantly improved(P<0.01).

CONCLUSIONRoux-en-Y gastric bypass can significantly improve the quality of life for non-obese patients with type 2 diabetes mellitus.

Adult ; Aged ; Diabetes Mellitus, Type 2 ; psychology ; surgery ; Female ; Gastric Bypass ; methods ; psychology ; Humans ; Laparoscopy ; methods ; psychology ; Male ; Middle Aged ; Obesity ; Quality of Life ; Retrospective Studies ; Surveys and Questionnaires ; Treatment Outcome