ABSTRACT
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.