SMAD4(Mothers against decapantaplegic homologue 4), a typical tumor supresor gene was initialy isolated from pancreatic ductal adenocarcinomas, which located on the chromosome 18q21.1. Mutation of SMAD4 gene found in human carcinomas such as pancreati...
SMAD4(Mothers against decapantaplegic homologue 4), a typical tumor supresor gene was initialy isolated from pancreatic ductal adenocarcinomas, which located on the chromosome 18q21.1. Mutation of SMAD4 gene found in human carcinomas such as pancreatic and colon carcinomas as wel as breast, esophageal and gastric carcinomas. Thus, many studies have focused on SMAD4 gene, especialy for the action mechanisms of oncogenesis and metastasis. This study aims to search novel SMAD4 binding partners by protein aray tols, and fluorescent or luminescent proteins and identify SMAD4 promotor activated on the human gastric carcinoma cel line and primary transcription factors that regulate SMAD4 promotor. In order to study novel SMAD4 binding partners, gateway cloning method was used for the human SMAD4, using the Baculovirus system was mas replication of SMAD4, and finaly purified SMAD4 protein obtained. The results of asay using purified SMAD4 protein were obtained for eighten diferent candidates through a new protein screning method, protoary. The four proteins were already known while the others were new thing and so validated by BiFC (Bimolecular Fluorescence Complementary) asay. The seven out of fourten proteins were identified as new interaction partners of SMAD4. Among them, functional study for thre of the proteins was performed. The interaction betwen FARN1 and SMAD4, and through this interaction, First, FARN1 negatively regulates SMAD4 in multiple levels including blocking SMAD4’s induction of p15INK4b and direct represion of Smad4 transcription and expresion. This founding could be a god indication of FARN1’s oncogenic role in cancer promotion. Second, F2ITE mRNA expresion was upregulated in human gastric cancer tisue and an inverse corelation exist betwen the mRNA levels of F2ITE and Smad4 in various gastric cancer cels. So, our finding showed first time the non-canonical role of F2ITE in the regulation of smad4, an important component of TGF-β signaling pathway. Finaly, we show that Smad4 interacts with a centrosomal oncogeni kinase KiAA. Our findings provide a mechanistic explanation for the recently reported role of KiAA as a tumor-susceptibilty protein in mouse and human. The function of KiAA as a Smad4 supresor makes it an important target for developing therapeutic strategies fothose human cancers in which KiAA is overexpresed. In order to study SMAD4 promoter, first, a deletion form of SMAD4 was employed to observe the basic activity on the gastric cel line. The location where the activation considerably decreased was detected. Second, a few candidate factors were found by the program established to search transcription factors on the site. Third, the transfection of candidate factors was performed on the gastric carcinoma cel to observe the expresion of SMAD4. Finaly, SSM factor was selected as a candidate that afects the expresion of SMAD4. Through Real-Time RT-PCR and western blot asay, it was shown that SSM increased the expresion of mRNA and protein of SMAD4. Also, luciferase asay was caried out to confirm that SSM increases the activation of SMAD4 promoter. By EMSA, it was shown that SSM binds to SMAD4 promoter, which the expresion of SMAD4 was increased by SSM on the gastric carcinoma cel line. Therefore, it was confirmed that SSM transcription factor on the gastric cel line binds to SMAD4 promoter to regulate SMAD4 expresion.