Objective To study the expression of MREll-RAD50-NBS1 complex in normal gallbladder tissues,simple cholecystitis,calculous cholecystitis and gallbladder carcinoma.Methods The expression of MRN complex in different gallbladder lesion tissues were detected by immunohistochemistry.Western blot,Methyl thiazolyl tetrazolium(MTT) assay and flow cytometry were used to detect the influence of apoptosis and proliferation induced by NBS1.Results The differences between the expression of MRE11,RAD50 and different gallbladder lesion tissues were not statistically significant (respectively x2 =2.724,1.697,all P ＞ 0.05).The expression of NBS1 in GBC tissues [15.3％ (9/59)] was prominently lower than that in the normal gallbladder tissues [84.7％ (50/59)],simple cholecystitis [87.8％ (36/41)],calculous cholecystitis [61.2％ (30/49)] (x2 =87.388,P ＜ 0.01).The Western blot results reveal that NBS1 can mediate apoptosis by up-regulate Bax and down-regulate Bcl-2.The MTT assay results showed that the relative absorbance of treatment and control group after 24,48,72 h were[(1.120 ± 0.006)vs.(1.350±0.009),(1.600±0.004)vs.(1.99±0.01),(1.83±0.01)vs.(2.260±0.003)(F=7.659,P ＜0.01).The apoptosis rate in treatment group(17.23％ ± 0.56％)was higher than that in the control group (4.13％ ± 0.67％) (t =9.133,P ＜ 0.01).Conclusion NBS1 is closely related to gallbladder carcinoma.NBS1 can inhibit the proliferation and promote apoptosis of GBC-SD cells.

BACKGROUND:Although asparagine synthetase (AsnS) is associated with drug resistance in leukemia, its function in extranodal natural killer (NK)/T-cell lymphoma (ENKTL) remains unclear.METHODS:The present study investigated the relationship between baseline AsnS mRNA levels and response to asparaginase in ENKTL cell lines. It also determined whether upregulating or downregulating the AsnS mRNA level induces or reverses asparaginase-resistant phenotype.RESULTS:Interestingly, considerable differences were observed in the sensitivity to asparaginase of the five ENKTL cell lines. The AsnS expression levels were positively correlated with the IC50 values. In addition, the asparaginase resistance was induced or reversed by upregulating or downregulating the AsnS mRNA level in vivo and in vitro. Functional analyses indicated that AsnS did not affect the proliferation and apoptosis of ENKTL cells in the absence of asparaginase.CONCLUSION:Together, the data stress the importance of AsnS in the sensitivity to asparaginase in ENKTL and suggest a different therapeutic strategy for patients with a different level of AsnS expression.

Objective Study on the mechanism of sliencing microRNA (miRNA,miR)-16 on chemoresistance in SMMC-7721.Methods The lentiviral vectors were used to construct stable cell lines.To determine whether constructed successfully,the expression of miR-16 was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and inhibitor of kappaB kinase beta (IKBKB),the target gene of miR-16,was detected by Western blotting.The sensitivity and resistance mechanism of the stable cell lines to paclitaxel were detected by Western blotting and thiazole blue (MTT).Results The expression of miR-16 in 7721-miR16 group was significantly higher than SMMC-7721 and 7721-mock groups (4.63 ± 1.32 vs.1.00 ± 0.30 vs.0.93 ± 0.37,P =0.000).The expression of IKBKB in 7721-shmiR16 was 25.73 times than the controls (P =0.000).The expression of multidrug resistance protein 1 (MDR1) in 7721-shmiR16 was increased and the half maximal inhibitory concentration (IC50) to paclitaxel was 4.903 μmol/L,10.64 times than the control group (0.461 μmol/L) (P =0.000).The results of western showed that silencing miR-16 induced resistance mainly through activating nuclear factor-kappaB (NF-κB) pathway.Conclusion Silencing miR-16 in SMMC-7721 significantly increases IKBKB expression leadind paclitaxel resistance.

Hepatocellular carcinoma (HCC) is a common malignant tumor usually resistant to chemotherapy. MicroRNAs play important roles in modulation of carcinogenesis and chemoresistance, which miR-16 has been reported to mediate chemoresistance in many types of cancers. However, the role of miR-16 in HCC remains unknown. The aim of this study was to investigate whether miR-16 is participated in chemoresistance in HCC and shed light on the underlying molecular mechanisms. The findings of the current study discover that miR-16 is down-regulated in HCC tissue and cell lines. The results demonstrate that the inhibition of miR-16 renders resistance to paclitaxel in vitro and in vivo by targeting IKBKB via NF-κB signaling pathway, suggesting that miR-16 may be a meaningful therapeutic potential to overcome drug resistance in HCC.

Objective To study the expressions of Nodal in normal gallbladder,and gallbladders with cholelithiasis,cholecystitis and carcinoma;and to study the impact of inhibiting or promoting Nodal expressions in gallbladder carcinoma on the Smad2/4 pathway and epithelial-mesenchymal transition.Methods Immunohistochemistry was used to detect the expressions and distributions of Nodal protein in 30 normal gallbladders,96 simple cholecystitis/calculous cholecystitis specimens and 42 gallbladder carcinoma specimens.The mRNA and protein expressions of Nodal in normal and malignant gallbladdcr mucosal epithelium cells and breast cancer cells were detected by RT-PCR,western blotting and wound healing tests.The impact of activating agents and inhibitors on the expression levels of Nodal and its signaling pathway Smad2/4 and EMT-related proteins were analyzed.Results Immunohistochemistry showed that the positive rates of Nodal in the gallbladder cancer group was significantly higher than that in the gallbladder stone group and the normal gallbladder group.The results were 83.3％ (35/42),44.8％ (43/96),6.7％ (2/30) respectively (P＜ 0.01).RT-PCR and Western blotting showed the expressions of Nodal in gallbladder carcinoma cells were higher than normal gallbladder cells (P＜0.05).After using rhNodal to up regulate the Nodal expression,the Smad2 protein phosphorylation was promoted and the EMT associated proteins were up-regulated.After using the inhibitor SB431542 to suppress the Nodal expression,the Smad2 protein phosphorylation decreased and the EMT associated proteins were down-regulated.Conclusions The expression of Nodal was closely related to cell proliferation and metastasis in gallbladder carcinoma.Tumor progression was promoted via the smad2/4 pathway through epithelial-mesenchymal transition.

Objective To investigate the expressions of Yes-associated protein-1 (YAP1) in gallbladder mucosal epithelium of normal persons,in patients with simple/calculous cholecystitis,and in patients with gallbladder carcinoma;and to study the mechanism of YAP1 in gallbladder carcinoma development.Methods Immunohistochemistry was used to detect the expression and distribution of YAP1 protein in 50 persons with normal gallbladder,101 patients with simple cholecystitis/calculous cholecystitis and 100 patients with gallbladder carcinoma.RT-PCR and western-blot were used to detect the mRNA and protein levels of YAP1 in normal and malignant gallbladder mucosal epithelium cells.siRNA was used to shut down the expression of YAP1 in SGC996 cells.MTT was used to test cell vitality.Flow cytometry was used to measure cell cycle.Results Immunohistochemistry revealed the expression rates of YAP1 in the gallbladder carcinoma group,the cholecystitis/gallstone group and the control group to be 87.0％ (87/100),56.4％ (57/101) and 5.0％ (1/20),respectively (P ＜ 0.01).The YAP1 protein levels were higher in gallbladder carcinoma tissues and cells when compared to normal tissues and cells.RT-PCR showed the mRNA levels of gallbladder carcinoma cells to be 12.5 ± 1.2 times of normal gallbladder mucosal epithelial cells (P ＜ 0.05).After using siRNA to shut down the YAP1 expression,EMT associated proteins were down-regulated,cell vitality was decreased,and cell cycle was arrested in the S-phase.Conclusions YAP1 is closely related to cell proliferation and metastasis of gallbladder carcinoma.It may promote tumor progression through epithelial-mesenchymal transition.transition;Tumor progress

Objective Hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to multidrug resistance (MDR). This study was designed to observe the effect of small interfering RNA (siRNA) targeting MDR1 in modulating drug resistance and apoptosis. Methods HCC MDR cell lines,SMMC-7721/adriamycin ( ADM), were developed by exposing parental cells to stepwise increasing concentrations of ADM from 0. 1 to 2. 0 mg/L. After siRNA (25, 50, 75 nmol/L) transfection MDR1 mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) at 24, 48, 72 h. methyl thiazol tetrazolium (MTT) assay was used to determine drug sensitivity 48 h after transfection. Flow cytometry (FCM) was employed to analyze cell apoptosis to ADM (0. 2mg/L). The expression of MDR1 protein was examined by Western blotting. Results MTT assay showed that the resistance index (RI) values of SMMC-7721/ADM cells against ADM, 5-Fu, VCR and oxaliplatin were 25.43, 68.32, 39. 17, 18. 31, and those after siRNA transfection were 5.01, 17.04, 4. 96, 1.62,respectively. QRT-PCR analysis demonstrated the MDR1 mRNA expression was decreased significantly in SMMC-7721/ADM cells after siRNA transfection (P＜ 0. 05 ). In addition, as compared with parental cells, MDR1 protein expression was apparently decreased in SMMC7721/ADM cells treated with siRNA (P ＜0. 05). Flow cytometry revealed significant apoptesis of the cells following siRNA transfection ( P ＜0. 05). Conclusion Inhibition of MDR1 by siRNA enhanced the selectively restored sensitivity to drugs.The MDR1 siRNA might represent a new therapeutic option for HCC.

In order to examine potential molecular markers of pancreatic stem cells, a regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in rat regeneration pancreas at 3rd day after Px, as comparing to sham surgery (Sx), were analyzed by using two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. HSP47 and Vimentin were analyzed in Px and Sx rat with Western blotting to confirm the results of 2DE. The average spots in gels for Sx and Px pancreatic tissues were (1 369 +/- 31) and (1 315 +/- 28). respectively. 2DE displayed 91 spots with at least 1.5-fold of differential expression at the time point of 3 days after pancreatectomy and 53 differentially expressed proteins were identified by PMF. Among them, Vimentin, CK8, L-plastin, hnRNP A2/B1 and AGAT are associated with embryogenesis and cells differentiation and may be new potential pancreatic stem cells markers. The proteome profiling technique provided a broad-based and effective approach for discovering potential biomarkers of pancreatic stem cells.