Visualizing P12^CDK2AP1-Upp Interactions In Living Cells By Bimolecular Fluorescence Complementation Assay And Biological Influence Of Exogenous Upp On SCC-25Cell

Oral cavity cancer is the sixth common one in the world. Histopathological studyshowed that most of these cancers are constituted of squamous cell carcinomas. Because itoften occurs on the head and neck， it seriously affects the patients’ physical and mentalhealth. The5-years survival for head and neck squamous cell carcinomas （HNSCCs） isamong the lowest of the major tumor types. With the development of molecular biology，directly against cancer pathogenesis， oncogene， tumor suppressor gene has become one ofthe main direction in gene therapy for cancer. CDK2AP1（cyclin dependent kinase2associatedprotein1）which has been suggested to be a candidate tumor suppressor gene was more and more popular. p12^CDK2AP1protein is its coding protein. p12^CDK2AP1affect the DNAreplication by regulating CDK2activity negatively. p12^CDK2AP1protein expression inHNSCCs is low or even none. p12^CDK2AP1protein maybe play an important oral in thetumorigenesis and developing. In2009we find a new binding protein Upp（unnamed proteinproducts） of p12^CDK2AP1protein by yeast two-hybrid technology. Upp chromosomal locates inhuman chromosome4. The Upp’s chromosomal location， sequence of nucleic acid andamino acid are similar with the MORF4（mortality factor on human chromosome4） protein.This study indicated p12^CDK2AP1–Upp interaction and biological influence of exogenous Uppon human tongue squamous cancer cells SCC-25.Objectives:1， To visualized p12CDK2-AP1–Upp interactions in living cells by bimolecular fluorescencecomplementation assay.2， To investigate the influence of biological functions of exogenous Upp on SCC-25cellline.Methods:1， We constructed the eukaryotic expression vectors pBiFC-VN173-P12andpBiFC-VC173-Upp by molecular clone technology.2， p12CDK2-AP1-Upp interactions in living cells were verified by bimolecular fluorescencecomplementation assay.3， The recombinant vector pBiFC-VC173-Upp was transfected into SCC-25cells. We useRT-qPCR and BiFC assays to measured the mRNA and protein expression of Upp. Weobserved the proliferation，invasion ability，cell cycle，apoptosis of the transfected SCC-25cell line in order to investigate the influence of biological functions of exogenous Upp onSCC-25cell line.Results:1， The eukaryotic expression vectors pBiFC-VN173-P12and pBiFC-VC173-Upp weresuccessfully constructed.2， Successfully verified the P12^CDK2AP1-Upp interactions and the subcellular localization in living cells by bimolecular fluorescence complementation assay.3， The proliferation and invasion of SCC-25cell line were negatively regulated byexogenous Upp. The percentage of cells in S phase was decreased and the percentage ofcell apoptosis was increased in the specific interference group.Conclusion：Bimolecular fluorescence complementation is a recently developed technique for detectionof protein-protein interactions in living cells. In this study， we have successfullyconstructed the eukaryotic expression vectors pBiFC-VN173-p12and pBiFC-VC173-Uppand verified the strong p12CDK2-AP1-Upp interactions fluorescence signals in living cells bythis assay. Exogenous Upp influence the proliferation，invasion，cell cycle，apoptosis ofSCC-25cells. Maybe Upp is a new tumor suppressor protein.