The Mechanism Of P12^CDK2-AP1 Binding Protein UPP In Neck And Head Carcinoma

Tumor is one of the severest of all kinds of human race diseases. It can seriousaffect human health and living. The malignant tumor （cancer） can threaten human lifeand quality of living， and can cause the mortal complications. So the tumourgenesis isalways the medical research important filed. Head and neck cancer share3%ofincidence rate，1.3%of mortality rate among the total tumors. The80-85%ofpathological types of head and neck cancer is squamous cell carcinoma. At present， inthe head and neck caner researching field， the relationship of oncology and cancersuppressor gene， the protein and protein interaction of key protein is regarded as one ofmain directions to be studied. CDK2-AP1（cyclin dependent kinase2associatingprotein1） gene is first discoveried in using a subtractive hybridization approach. It wasidentified as tumor suppressor gene that is structurally altered during oral carcinogenesisand expressed only by normal and not by transformed hamster oral keratinocytes. Thep12^CDK2-AP1protein is coded by CDK2-AP1gene. The mechanism of p12^CDK2-AP1caninhibit cancer proliferation and phenotype. But the mechanism is not clearly.Objectives:1. To identify the interaction of p12^CDK2-AP1and the novel protein UPP through pull-downand co-IP assays.2. Using the p12^CDK2-AP1siRNA and overexpression to investigate the expression of UPPgene. 3. Using stably infected TCA-8113-UPP to study mechanism of UPP gene in vivo.Methods:1. The pGEX4T-p12^CDK2-AP1and pGEX4T-UPP plasmids were transformed into BL21E. coil to express GST-p12^CDK2-AP1and GST-UPP fusion proteins. Theisopropyl-beta-thiogalactopyranoside （IPTG）200ul1mM was added into the flasksand induced for5hours. The cells were harvested with PBS. Pelleted cells were lysedby sonication in PBS containing1%Triton X-100and complete protease inhibitorcocktail （Roche）. After centrifugation, supernatants were applied toglutathione–Sepharose4B. The thrombin protease （GE healthcare, USA） was addedinto the GST-UPP tube. The beads were pelleted, washed three times in bindingbuffer, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE.2. The pCDNA-HA-p12^CDK2-AP1and pCDNA-FlAG-UPP plasmids were used to performthe Co-IP assay according to the user manual. The293T cells were lysed inimmunoprecipitaion （IP） buffer after transfected for forty-eight hours. Lysate wasdivided into two spin columns respectively. The samples were analyzed by SDS-PAGE.Proteins were transferred onto nitrocellulose membranes and then incubated withprimary antibodies. The anti-HA antibody was added onto UPP membrane while theanti-FLAG antibody onto the p12^CDK2-AP1membrane. After washing4times withTBST, the membranes were subsequently incubated with horseradish peroxidaseconjugated secondary antibodies. The ECL was used to detect horseradish peroxidaseon immunoblots. The dried membranes were exposed to Kodak Biomax MR film atroom temperature.3. The p12^CDK2-AP1siRNA oligo was designed to perform the siRNA assay. Aftertransfection, the mRNA of UPP gene was extracted and measured by quantitativereal-time PCR.4. Through p12^CDK2-AP1gene transfected in293T cell line, the quantitative real-timePCR was used to verify the expression of UPP gene.5. The lentivirus overexpression vector pCDH-GFP-MCS was constracted to infect the TCA-8113cells. The positive clone of the stable infection UPP gene cells werescreened out by flow cytometry. The stably infected TCA-8113-UPP gene cells weresubcutaneously injected into the nude mice. After the tumors exceed the1-3mm3,thetumors were harvested to weight and calculate.Results:1. P12CDK2-AP1and UPP have the direct interaction in vitro and in vivo.2. CDK2-AP1can regulate the expression of UPP gene.3. The UPP gene can decrease of G2/M phase of cell cycle.4. The UPP gene can inhibit proliferation to human tumor xenografts in vivo.Conclusions:1. In this study,we verified that the novel protein UPP can interact with p12^CDK2-AP1.2. P12CDK2-AP1can mediated the expression of UPP gene, the UPP may locate thedownstream of p12^CDK2-AP1. P12CDK2-AP1exert its mechanism by interacting with UPP.3. Our study showed that The UPP gene can inhibit the proliferation of tumor.