ABSTRACT
Background and purpose:DOC-2 serves as one of the tumor suppressing genes in human ovarian cancer and plays a role in the process of cell growth and differentiation.This study was to investigate the role of DOC-2 in the TGF? signal pathway and verification of the interaction between DOC-2 and TGF?Ⅲ receptor.Methods:The bait vector was constructed by inserting the PID domain of DOC-2(nDOC-2)into yeast express vector pGBKT7.pGBKT7-nDOC2 was transformed into the yeast AH109 and confirmed to be expressed.After the human foetus brain cDNA library had been transformed,the positive clones was screened by both nutrition defect medium and X-?-gal.The putative positive clones were sequenced and analyzed to get the DOC-2 interactive proteins.Furthermore,after the DOC-2 cDNA and TGF?Ⅲ receptor cDNA had been co-transfected into the human ovarian cancer cell line HO-8910 together,the interaction between DOC-2 and TGF?Ⅲ receptor was investigated by immunoprecipitation and Western blot.Results:21 putative positive clones were picked after being screened and sequenced.Three of them were identified as Homo sapiens partial mRNA for betaglycan(TBR Ⅲ gene),homo sapiens protocadherin gamma subfamily C3(PCDHGC3)and APLP1(amyloid beta precursor-like protein 1).The analysis by immunoprecipitation and Western blot showed that the interaction between DOC-2 and TGF?Ⅲ receptor could form protein complex.Conclusions:The three encoding proteins might participate in the DOC-2 signal pathway.DOC-2 might play as an essential role in the TGF?signal pathway by interacting with TGF?Ⅲ receptor.
ABSTRACT
Objective: To screen for proteins which can interact with phosphotyrosine-interacting domain (PID) of differentially expressed gene in human ovarian cancer cell line DOC-2 by yeast-two hybrid technique, so as to provide evidence for the signal pathway of DOC-2. Methods: The cDNA sequence of human DOC-2 gene was amplified and its PID domain (nDOC-2) was subcloned into the bait vector pGBKT7 of yeast two-hybrid system; the product was then used to screen an embryo brain cDNA library and the proteins interacting with nDOC-2 were identified. Quadrople dropout(QDO) medium and X-?-gal were used for selecting the positive clones. PCA was used to analyze the amplified sequence. After elimination of the false positive clones, the positive clones were sequenced and analyzed by bioinformatic methods. Results:Twenty-one candidate positive clones were obtained and 3 of them were plasmids encoding Homo sapiens partial mRNA for betaglycan (TBR III gene), Homo sapiens protocadherin gamma subfamily C 3 (PCDHGC3), and APLP1(amyloid beta precursor-like protein 1).Conclusion: The proteins obtained in this study may play important roles in the signal pathway of DOC-2, which provides a new orientation for DOC-2 gene therapy of ovarian cancers