학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2012. 8. 신영기.GNAI2 (Guanine nucleotide binding protein (G-protein), alpha inhibiting activity polypeptide 2) belongs to a family of Gi alpha proteins that includes three
polypeptides: Gi alpha 1 (GNAI1), Gi alpha 2 (GNAI2), and Gi alpha 3 (GNAI3). GNAI2 is a well known proto-oncogene that is involved in onset and propagation of
many different types of cancers. Smad4 (Mother Against Decapentaplegic Homologue 4, DPC4) is a well-known tumor suppressor and key elements in TGF-β (Transforming Growth factor- β) signaling pathway. In order to demonstrate the role of GNAI2 in carcinogenesis and to find out its potential interacting partners, we had initially identified Smad4 as one of its interacting partners through a baculovirus
protoarray system. In this study we firstly demonstrated the novel interaction between GNAI2 and Smad4 using bimolecular fluorescence complementation
(BiFC) assay in cervical cancer (HeLa) cell. We also confirmed the interaction between GNAI2 and Smad4 by endogenous and exogenous immunoprecipitaion (IP)
assay. Upon ectopic transfection of GNAI2, the western blot analysis resulted in decrements of the level of Smad4, p15INK4b, p21 protein and an increase the
protein expression of C-Myc. In contrast, transient knockdown of GNAI2 resulted in substantial increase in Smad4, p15INK4b, p21 protein and a decrease in the protein expression of C-Myc. However, our qRT-PCR data showed no change at mRNA level of Smad4 but a substantial decrease and increase at mRNA level of p15INK4, p21 after transient overexpression and knockdown of GNAI2 in HeLa cell respectively. Moreover, 26S proteosomal degradation inhibitor MG132 treatment in
GNAI2 overexpressed HeLa cell resulted in reduced degradation of Smad4 protein.Through luciferase reporter assay we have found that the overexpression of GNAI2 resulted in reduced p15INK4b promoter activity. In addition, the si-RNA-based knockdown of GNAI2 significantly reduced the proliferation of cervical cancer (HeLa) cells. Taken together our study demonstrated that GNAI2 interacts with Smad4 and negatively regulates the expression of Smad4 and its targeted gene in cervical cancer (HeLa) cells.Elucidate the role GNAI2 in the regulation of Smad4 function and its consequences in cervical carcinogenesis.Maste
