Effects of Mucin 3A silencing by shRNA mediated with lentivirus vector on the growth of human extrahepatic cholangiocarcinoma / 中华肝胆外科杂志

Objective To investigate the effect of Mucin 3A (Muc3A) gene silencing by shRNA mediated with lentivirus vector on human extrahepatic cholangiocarcinoma.Methods Short hairpin RNA (shRNA) interference sequence targeting Muc3A gene was designed and synthesized.Recombinant lentiviral plasmids were packaged to produce virus venom and their titers were determined.After transfected with QBC939 cells of extrahepatic cholangiocarcinoma,stable positive cell lines were obtained by optimal drug screening concentration.QBC939 cells were divided into three groups:lentivirus mediated shRNA transfected cells (transfected group),empty virus transfected cells (negative control group),and untransfected cells (blank group).ShRNA silencing efficiency of Muc3A gene was detected with Western blot.Cell growth was assessed by MTS assay,cell colony formation was detected with plate clonogenesis assay,and cell cycle distribution were detected by flow cytometry.Results Lentivirus was successfully packaged and titer of virus suspension was 1 × 108 TU/ml.Western blot confirmed that shRNA worked well in QBC939 cells.3 μg/ml puromycin concentration was added for table cell lines selection.Western blot results showed that the expression of Muc3A in the transfection group was significantly decreased comparing with negative control group and the blank group (P ＜ 0.05).The MTS results showed that the value of OD490nm in the transfected group was significantly lower than that in the negative control group and the blank group,and the differences were statistically significant (P ＜ 0.05).The number of clone formation in the transfection group was significantly lower than that in the negative control group and the blank control group,and the differences were statistically significant (P ＜ 0.05).Cell cycle of the experimental group was in G2/M is more,but S phase is less,but there is no statistical difference compared with blank group (P ＞ 0.05).Conclusion Lentivirus mediated shRNA transfection can significantly inhibit the growth,proliferation and colony formation of QBC939 cells of extrahepatic cholangiocarcinoma after interfering with Muc3A gene expression,which sugests that Muc3A can promote the growth of cholangiocarcinoma cells.

MUC Expression in Gallbladder Epithelial Tissues in Cholesterol-Associated Gallbladder Disease

BACKGROUND/AIMS: Gallstone pathogenesis is linked to mucin hypersecretion and bacterial infection. Several mucin genes have been identified in gallbladder epithelial cells (GBECs). We investigated MUC expression in cholesterol-associated gallbladder disease and evaluated the relationship between mucin and bacterial infection. METHODS: The present study involved 20 patients with cholesterol stones with cholecystitis, five with cholesterol stones with cholesterolosis, six with cholesterol polyps, two with gallbladder cancer, and six controls. Canine GBECs treated with lipopolysaccharide were also studied. MUC3, MUC5AC, MUC5B, and MUC6 antibodies were used for dot/slot immunoblotting and immunohistochemical studies of the gallbladder epithelial tissues, canine GBECs, and bile. Reverse-transcription polymerase chain reaction was performed to evaluate MUC3 and MUC5B expression. RESULTS: MUC3, MUC5AC, MUC5B, and MUC6 were expressed in the normal gallbladder epithelium, and of those, MUC3 and MUC5B exhibited the highest expression levels. Greatly increased levels of MUC3 and MUC5B expression were observed in the cholesterol stone group, and slightly increased levels were observed in the cholesterol polyp group; MUC3 and MUC5B mRNA was also upregulated in those groups. Canine GBECs treated with lipopolysaccharide also showed upregulation of MUC3 and MUC5B. CONCLUSIONS: The mucin genes with the highest expression levels in gallbladder tissue in cholesterol-associated diseases were MUC3 and MUC5B. Cholesterol stones and gallbladder infections were associated with increased MUC3 and MUC5B expression.

Expression of MUC3, MUC5AC, MUC6 and Epidermal Growth Factor Receptor in Gallbladder Epithelium according to Gallstone Composition / 대한소화기학회지

BACKGROUND/AIMS: Gallbladder (GB) mucin is one of the key factors in the gallstone formation. However, there is little information about the diversity of mucin secretion according to the stone composition. Epidermal growth factor receptor (EGFR) functions in proliferation including mucin secreting goblet cell hyperplasia. We compared the expressions of MUC3, MUC5AC, MUC6 and EGFR in the GB epithelium with cholesterol gallstones (GB-chol) group and pigment gallstones (GB-pig group). METHODS: GBs from elective laparoscopic cholecystectomy for the gallstone disease were studied. Stone composition was analyzed by the spectrophotometer. Immunohistochemical stain was performed using each monoclonal antibody. The percentage of stained proportion was scored by the NIH image program and the results were compared between both groups. RESULTS: Total 20 patients were enrolled (10 patients with cholesterol gallstones, 10 patients with pigment gallstones). The percentages of stained proportion for MUC3, MUC5AC, and MUC6 were 42+/-27%, 31+/-15%, and 17+/-9%, respectively in GB-chol group and 32+/-22%, 33+/-23%, and 15+/-10%, respectively in GB-pig group (p>0.05). The expression of EGFR was 50% (5/10) in the GB-chol group and 80% (8/10) in the GB-pig group respectively. CONCLUSIONS: There was no difference in the expressions of MUC3, MUC5AC, and MUC6 between the two groups. Further studies are needed to elucidate the role of EGFR in the gallstore formation.