Study On The Application Of Rap2b SiRNA In Anticancer Therapy And Its Related Molecular Mechanism

Rap2b,a member of the RAS-related protein family,is a newly discovered downstream target of p53.When p53+/+ cells are exposed to DNA damage agents such as adriamycin,p53 is activated and induces the expression of Rap2b,which promotes cell survival.Therefore,we speculate that Rap2b is an effective anticancer target.In this study,we designed a gold nanoshell-based drug/gene delivery system to evaluate the potential of Rap2b siRNA as an anticancer therapeutic agent.In the meanwhile,we studied the interaction between Rap2b and p53 to provide clues for deciphering the anticancer mechanism of Rap2b siRNA.Firstly,we prepared the Rap2b monoclonal antibody for subsequent experiments.We obtained eight stable cell lines of monoclonal antibody.The results of ELISA showed subtype of eight antibodies were IgGl.At the same time,the results of western blot showed the minimum concentration of antibody was 0.1 μg/mL and the minimum detectable protein level was 5 ng.Secondly,the gold nanoshell particles were synthesized,and the synthesized Rap2b siRNA and adriamycin were coupled to the nanoparticles.After incubation with HCT116 cells,the adriamycin or Rap2b siRNA coupled with nanoparticles were internalized into the cells,which could be visualized under a confocal laser scanning microscope.Subsequent laser irradiation promoted the release of adriamycin and Rap2b siRNA from the nanoparticles and the released Rap2b siRNA decreased cellular expression of Rap2b.Next,we evaluated the anticancer effect of different combination of adriamycin,Rap2b siRNA,and their nanoshell derivatives in vitro and in vivo.The results showed the decreased expression of Rap2b significantly enhanced the anticancer effect of adriamycin.In addition,laser irradiation of the nanoparticles might exert an additional thermal killing effect on cancer cells and further improved the anticancer efficacy of adriamycin.Finally,an interaction between Rap2b and p53 was studied by GST pulldown and bimolecular fluorescence complementation assays.The data demonstrated that Rap2b could bind to p53 directly in vitro and in vivo.In order to further decipher the regions in these two proteins mediating the interaction,we constructed a series of Rap2b and p53 truncated mutants and again peroformed the GST pulldown and bimolecular fluorescence complementation assays.The data indicated that the 41-135 amino acid residues of Rap2b and the DNA binding domain of p53 are critical regions mediating the Rap2b-p53 interaction.In summary,our data demonstrated that Rap2b siRNA could significantly enhance the anticancer effect of adriamycin and Rap2b could bind to p53 directly,indicating that Rap2b might negatively regulate p53 activity by physically interacting with p53.These findings will help to understand the role of Rap2b and p53 in tumorigenesis and drug resistance and provide new targets for the development of anti-tumor drugs,and thus has an important significance for prevention and treatment of cancers.