ABSTRACT
Constitutively activated signal transducer and activator of transcription 3 (STAT3) plays an important role in the formation of many tumors including ovarian cancer. In this study, RNA interference specific to STAT3 was employed to study its effects on the inhibition of STAT3 signaling and on the growth of ovarian cancer CAOV3 cells. Plasmid vectors pGenesil-1-GFP-U6 expressing specific small hairpin RNA (shRNA) against STAT3 and the scrambled shRNA control were constructed. After transfection into CAOV3 cells, the STAT3 shRNA specifically suppressed STAT3 expression at both mRNA and protein levels. At the same time, expressions of Bcl-xL, cyclin D1, and c-myc were down-regulated, whereas the cleaved caspase 3 was up-regulated. In addition, STAT3 knockdown inhibited anchorage-independent growth and induced apoptosis in CAOV3 cells, and decreased tumor growth in nude mice implanted with ovarian cancer cells.
Subject(s)
Genetic Therapy/methods , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/therapy , STAT3 Transcription Factor/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Cell Survival , Female , Gene Silencing , Humans , Male , Mice , Mice, Nude , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/therapeutic use , Treatment OutcomeABSTRACT
Gab2 plays an important role in FcepsilonRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 (scaffolding adapter Grb2-associated binder 2) siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2-mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl (DNP)-specific IgE overnight and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of beta-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine, reduced the production of IL-4 and TNF-alpha and inhibited the phosphorylation of Akt, PKCdelta and p38 mitogen-activated protein kinase (MAPK). Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the FcepsilonRI-mediated Ca(2+)-independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated.
Subject(s)
Antigens/immunology , Mast Cells/immunology , Phosphoproteins/metabolism , RNA, Small Interfering/metabolism , Adaptor Proteins, Signal Transducing , Animals , Calcium/pharmacology , Calcium Signaling/drug effects , Cell Degranulation/drug effects , Cell Line , Cytokines/biosynthesis , Gene Expression Regulation/drug effects , Histamine Release/drug effects , Mast Cells/drug effects , Mast Cells/enzymology , Mast Cells/physiology , Phosphoproteins/genetics , Phosphorylation/drug effects , Rats , Receptors, IgE/immunology , beta-N-Acetylhexosaminidases/metabolismABSTRACT
AIM: To assess the effect of gender on genetic polymorphism of cytochrome CYP2C19 in Chinese population. METHODS: The genetic polymorphism of 140 healthy Chinese were analysed by PCR-RFLP (restriction fragment length polymorphism). RESULTS: Of 52 genotyped male subjects, 23 (44.23%) were homozygous for wildtype (wt/wt), 6 (11.54%) were homozygous for CYP2C19 m1 (m1/m1), and 23 (44.23%) were heterozygous for CYP2C19 m1 or CYP2C19 m2 (wt/m1 or wt/m2); and among the 88 genotyped female subjects, 31 (35.23%) were homozygous for wildtype (wt/wt), 13 (14.82%) were homozygous for CYP2C19 m1 (m1/m1), and 44 (50.0%) were heterozygous for CYP2C19 m1 or CYP2C19 m2 (wt/m1 or wt/m2); no homozygous genotype for CYP2C19 m2 (m2/m2) was found in the study. CONCLUSION: There is no statistical difference in ocurance of wt/wt and m1/m1 between in male and in female, so gender have no significant effect on genetic polymorphism of cytochrome CYP2C19.
