The Effect Of P12^CDK2-AP1 Interacting With CD82 On The Oral Squamous Cell Carcinoma Cells OSCC-15 And The Transplanted Tumor In Nude Mice

Protein interaction plays a pivotal role in the control of tumor biological processes. Our team cooperates with doctor Shao-Wu Zhang and Chen Wei who belong to College of Automation, Northwestern Polytechnical University. For investigating the p^12CDK2-AP1 role in cell cycle regulation and tumorigenesis, we used the decision templates based on multiple classier fusion and PPI-PKSVM to predict that p^12CDK2-AP1 interacts with CD82, and then, we prove that p^12CDK2-AP1 can interact with CD82 with Co-IP and the transplanted tumor in nude mice.Objectives:1. We used the computational methods to predict and prove the interaction proteins of p^12CDK2-AP1.2. To identify the embryo interaction of p^12CDK2-AP1 and the protein CD82 through Co-IP assays.3. To investigate the influence of biological functions of p^12CDK2-AP1 interacting with CD82 on OSCC-15 cell line.4. To study the mechanism of p^12CDK2-AP1 interacting with CD82 in vivo with the transplanted tumor in nude mice.Methods:1. We predictd and proved the interactions of p^12CDK2-AP1 using the decision templates based on multiple classier fusion and PPI-PKSVM.2. We constructed the eukaryotic expression vectors p IRES2-EGFP-p^12CDK2-AP1 and p IRES2-EGFP-CD82 by molecular clone technology.3. After sequencing, plasmids were transfected into the 293 T and OSCC-15 cells. The protein levels of p^12CDK2-AP1 and CD82 were examined using Western blot at 0 h, 24 h, 48 h and 72 h after transfection. Co-IP analysis was used to identify whether p^12CDK2-AP1 and CD82 could interact with another in eukaryotic cells.4. We next investigated the effects of p^12CDK2-AP1 and CD82 overexpression the potential influences of p^12CDK2-AP1 and CD82 on the growth with MTT;on the invasion capability of OSCC-15 cells in vitro using a Transwell system; to assess the potential role of p^12CDK2-AP1 and CD82 on cell apoptosis with Annexin-V-FITC and PI.5. To understand the influence of p^12CDK2-AP1 and CD82 in vivo tumor formation, non-transfected cells（OSCC-15）, cells transfected with NC plasmid（OSCC-15/NC）, or p^12CDK2-AP1 plus CD82(p^12CDK2-AP1/CD82) plasmid were subcutaneously inoculated into nude mice. The tumor mass were measured using a precision caliper every three days initiated on Day 7 and ending on Day 25,then the tumor size and the tumor inhibitory rate were calculated.Results:1. Using the multiple and pairwise kernel support vector machine, 10 proteins were predicted to have high potential in binding with p^12CDK2-AP1. Among these proteins, tumor suppressor gene CD82 were chosen for further analysis. The sequences of p^12CDK2-AP1 and CD82 were input into the pairwise kernel support vector machine, and the two proteins were predicted as an interacting protein-protein pair. And we chose CD82 as an interacting protein-protein for the study.2. The pIRES2-EGFP-p^12CDK2-AP1 and pIRES2-EGFP-CD82 recombinant plasmids were constructed successfully.3. The p IRES2-EGFP-p^12CDK2-AP1 and p IRES2-EGFP-CD82 recombinant plasmids were overexpressed in 293 T cells. Co-immunoprecipitation was conducted to validate the direct binding between p^12CDK2-AP1 and CD82.4. OSCC-15 cells transfected with NC, p IRES2-EGFP-p^12CDK2-AP1 and p IRES2-EGFP-CD82 alone or in combination, the combined transfection of both plasmids was significantly more efficient in inhibiting the proliferation and cell invasion of OSCC-15 cells, but promoting cell apoptosis compared with the other groups.5. The combined overexpression of p^12CDK2-AP1 and CD82 inhibits the growth of OSCC-15 cells in tumor mouse xenografts.Conclusion:We constructed p IRES2-EGFP-p^12CDK2-AP1 and p IRES2-EGFP-CD82 recombinant plasmids. Our study revealed that when p^12CDK2-AP1 and CD82 were co-expressed in OSCC-15 cells, a synergistic tumor inhibition in the proliferation and cell invasion and promoting cell apoptosis were detected both in vitro. Our study also found that the combined overexpression of p^12CDK2-AP1 and CD82 inhibited the growth of OSCC-15 cells in vivo.