【Abstract】ObjectiveTo investigate whether abnormal expression of β-catenin and high expression of c-myc have played a possible role in hilar cholangiocarcinoma carcinogenesis.MethodsBy using immunohitochemical staining (SP method), the authors detected the expression of β-catenin and c-myc in 42 paraffin-embedded samples of hilar cholangiocarcinoma and 10 benign bile duct disease tissue, and then analyzed the relationship of them with clinical data. Resultsβ-catenin was normally expressed in 10 benign bile duct disease tissue, while expression of c-myc was negtive. In hilar cholangiocarcinoma tissue, the positive expression rate of β-catenin （71.4%） was significantly correlated to the lymphoid node metastasis of hilar cholangiocarcinoma (χ2=4.75，P＜0.05),but was not statistically correlated to the tumor size，the extent of differentiation and infiltration (χ2=3.35，3.45，4.32，Pgt;0.05); the expression rate of c-myc (76.2%) was correlated with the extent of differentiation(χ2=4.87, P＜0.05),but not with the size, infiltration, lymphoid metastasis（χ2= 3.47，4.12，2.76， Pgt;0.05). The abnormal expression of β-catenin had relevance to the high expression of c-myc with hilar cholangiocarcinoma （r=0.324,P＜0.01）. ConclusionThe expression of beta-catenin and c-myc is significantly altered in hilar cholangiocarcinoma, and correlate with biological features of cholangiocarcinoma.The abnormal expression of beta-catenin is one of the mechanisms for the spread of hilar cholangiocarcinoma.
ObjectiveTo investigate the effects of somatostatin8 (SS8) on the apoptosis and the expression of cmyc protein of hepatocellular carcinoma cell SMMC7721. MethodsCultured in vitro, hepatocellular carcinoma cells SMMC7721 were incubated with SS8 (10 μg/ml). The apoptosis rate and expression of cmyc protein were detected by flow cytometry (FCM). ResultsSS8 can cause the spanonumber in S and G2/M phase and the auxonumber in G0/G1 phase of SMMC7721 cells . The apoptosis rate was 14.2% in the study group and 6.1% in the control group, and there was significant difference (P＜0.05); The level of expressions of cmyc protein was 0.833±0.035 after action by SS8 for 24 h. Compared with control group, there was no significant difference in the study group（P＞0.10）.But after the cells were incubated with SS8 for 48，72，96，120，144 h, the level of expressions of cmyc protein was 0.818±0.04，0.721±0.029，0.669±0.026，0.648±0.045，0.642±0.028 respectively in the study group, and there was significant difference as compared with the control group (P＜0.05). Conclusion The SS8 can induce the apoptosis and lower expression of cmyc protein of hepatocellular carcinoma cell SMMC7721.
Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
Objective To determine whether local delivery of c-myc shRNA could inhibit hyperplasia and lithogenic potentiality in a rat model of chronic proliferative cholangitis (CPC) via specific blockade of the c-myc expression. Methods The CPC animal model (CPC group) was established via retrograde insertion of a 5-0 nylon thread into the common bile duct through Vater’s papilla. Three kinds of c-myc shRNAs were then respectively injected in c-myc shRNA group, which were included shRNA-1, shRNA-2, and shRNA-3, respectively. Negative control group and sham operation group were established for comparison. Subsequently, histopathological changes of bile duct wall were observed by HE, Massion, and PAS/AB staining; c-myc protein was detected by immunohistochemistry method; 5-bromodeoxyuridine (BrdU) protein was tested by immumofluorescence method; c-myc, Mucin 3, and Procollagen Ⅰ mRNAs were detected by real time PCR; Ki-67 protein was determined by Western blot; Activity of β-glucuronidase was measured by modified Fisherman method. Results ①Compared with the CPC and negative control groups, biliary tract mucosa epithelium (HE staining), submucosal acid mucinous gland (mid-blue staining, PAS/AB staining), and degree of over-hyperplasia of collagen fiber in bile duct wall (blue staining, Massion staining) were weaker in the c-myc shRNA group. ②The expressions of c-myc mRNA, Mucin 3 mRNA, Procollagen Ⅰ mRNA, Ki-67 protein, and β-G activity in the c-myc shRNA group were lower than those of the CPC and negative control groups (Plt;0.05), but higher than those of the sham operation group (Plt;0.05). Conclusion c-myc shRNA treatment could effectively inhibit the hyperplastic behavior and lithogenic potential of CPC, which might help to prevent the biliary restenosis and stone recurrence.
Objective Hepatitis B virus X (HBx) protein is involved in the initiation and progression of hepatocellular carcinoma (HCC) by regulating the host protein-coding genes. Herein, we want to explore whether HBx protein can alter the expression of microRNAs (miRNAs) to promote proliferation and transformation in malignant hepatocytesin vitro. Methods MiRNA microarray and quantitative reverse-transcription polymerase chain reactions (qRT-PCRs) were performed to identify miRNAs that were differentially regulated by HBx protein in HCC cells. Protein and mRNA expression analyses, cell cycle and apoptosis analyses, and luciferase reporter assays were performed to delineate the consequences of miR-16 family repression in HepG2 cells. Results HBx protein induced widespread deregulation of miRNAs in HepG2 cells, and the downregulation of the miR-16 family was reproducible in HepG2, SK-HEP-1, and Huh7 cells. CCND1, a target gene of the miR-16 family, was derepressed by HBx protein in HepG2 cells. C-myc mediated the HBx-induced repression of miR-15a/16 in HepG2 cells. Ectopically expressed miR-15a/16 suppressed the proliferation, clonogenicity, and anchorage-independent growth of HBx-expressing HepG2 cells by arresting them in the G1 phase and inducing apoptosis, whereas reduced expression of miR-16 accelerated the growth and cell-cycle progression of HepG2 cells. Conclusions HBx protein altered thein vitro expression of miRNAs in host malignant hepatocytes, particularly downregulating the miR-16 family. Repression of miR-15a/16 is c-myc mediated and is required for the HBx-induced transformation of HepG2 cellsin vitro. Therefore, miR-16 family may serve as a therapeutic target for hepatitis B virus (HBV)-associated HCC.
ObjectiveTo investigate the effect of curcumin on the expression regulation of endogenousβ-glucoronidase (β-GD) induced by lipopolysaccharide (LPS).Methods① Human normal intrahepatic biliary epithelial cell line (HiBEpiC) cells in the logarithmic growth phase were divided into blank control group (0 h group) and 7 different stimulation time groups. The cell density was adjusted to 1×104/mL, and the cells were stimulated with 100 mg/mL LPS for 1, 3, 6, 18, and 24 hours respectively, including another two groups where the cells were cultured with LPS-free medium for 18 and 24 hours after LPS stimulation for 24 h. ② HiBEpiC cells in the logarithmic growth phase were divided into blank control group, LPS+low, medium, and high concentration curcumin group. The cell density was adjusted to 1×104/mL. In the blank control group, cells were not stimulated with any reagent; in the LPS group, cells were stimulated with 100 mg/mL LPS, in the other three groups, the cells were stimulated with 100 mg/mL LPS and simultaneously 20, 40, and 80 μmol/L curcumin, respectively, for 24 hours. The expressions of c-myc and endogenous β-GD were detected by Western blot method.Results① The expressions of endogenous β-GD and c-myc in HiBEpiC cells gradually increased with the prolongation of treatment time by LPS, and the expression levels of β-GD and c-myc at each time point group were significantly different from those in the 0 h group (P<0.05). ② There were significant difference between any two groups of the blank control group, LPS group, LPS+low concentration of curcumin group, LPS+medium concentration of curcumin group, and LPS+high concentration of curcumin group (P<0.05).ConclusionCurcumin is able to inhibit the increased expression of endogenous β-GD induced by LPS, possibly via inhibiting expression of c-myc.
Cholangiocarcinoma; Vascular endothelial growth factor; c-myc gene
Objective To investigate the effect on expression of c-myc and proliferating cell nuclear antigen （PCNA） of vein grafts transferred by c-myc antisense oligodeoxynucleotides（ODN） of soluble stent. Methods A rabbit model of common carotid arteries grafted by external jugular veins was constructed in 50 New Zealand rabbits and were randomly divided into five groups, 10 rabbits each group. Control group： no stents ; group 1： soluble stent ; group 2： soluble stent with sense-ODN; group 3： soluble stent with antisense-ODN; group 4.. soluble stent with mismatch-ODN. At 7 d, 28 d and 90 d after surgery, vein grafts were harvested. The expression of c-myc and PCNA were identified by immunochemistry methods. Results At 7d, 28d, 90d after surgery, the expression of c-myc and PCNA of the intima and media of vein grafts in control group, group 1, group 2, group 4 were higher significantly than that in group 3 （P〈0. 01）. At 28d, 90d after surgery, the expression of c-myc in five groups were higher than that in the same group at 7d after surgery （P〈0. 01）. Conclusion Soluble stent can transfer ODN effectively. C- myc antisense-ODN transferred by soluble stent can inhibit significantly the expression of c-myc and PCNA in the intima and media of vein grafts.