Effect Of CDK4 Protein Expression On Subcellular Localization Of P16 In Breast Cancer

Background As a tumor biomarker,p16 had been used for clinical diagnosis in many aspects,and the cytoplasmic translocation of p16 protein was associated with malignant tumors and its poor prognosis.In our previous study,our group confirmed that the subcellular localization of CDK4 protein and p16 protein in breast cancer tissues is highly consistent,mainly in the cytoplasm or both in the cytoplasm and nuclear.At the same time,studies had shown that the repeat sequences of ankyrin of p16 protein and the R24 site of CDK4 bind through electrostatic attraction,and p16/CDK4 complex existed both in the nucleus and in the cytoplasm.We hypothesized that the cytoplasmic translocation of p16 protein in breast cancer correlates with the formation of the p16/CDK4 complex in the cytoplasm of breast cancer,most likely due to the fact that CDK4 formed p16/CDK4 complex in the cytoplasm that renders the p16 protein inaccessible to the nucleus,the expression of p16 protein in the cytoplasm is up-regulated.Therefore,overexpression of CDK4 protein,expression of the mutant at the R24 site,and the knockdown of CDK4 protein were made respectively in BT549 cells.After that,in order to explore the regulatory mechanism,immunofluorescence and western blot were used to observe the subcellular localization of CDK4 and p16 protein.Purpose Explore the effect of different states of CDK4 protein on the subcellular localization of p16 protein in tumor cells,and then analyze the localization mechanism of p16 protein in the cytoplasm and nuclear.Methods1.Subcellular localization of CDK4 and p16 proteins in BT549 and MCF7 cell lines was observed by immunofluorescence.2.Select plasmids to knockdown CDK4 as much as possible via Real-time RT-PCR and western blot(pGPU6-sh1,pGPU6-sh2,pGPU6-sh3,pGPU6-sh4 were constructed and synthesized by the Jima company).3.Construct cells to stably express pEGFP-CDK4,pEGFP-CDK4R24 C and knockdown CDK4(pEGFP-CDK4,pEGFP-CDK4R24 C were constructed and synthesized by the General Biosysterns company).4.The method of immunofluorescence was used to observe the subcellular localization of CDK4 and p16 protein.Results1.Results of immunofluorescence showed that the p16 protein of BT549 and MCF7 located mainly in the cytoplasm or both in the cytoplasm and nucleus,and CDK4 protein located both in the cytoplasm and nucleus.The co-localization of two proteins existed.2.Results of Real-time RT-PCR and western blot showed that pGPU6-sh1 and pGPU6-sh4 had the obvious effect to knockdown CDK4 protein.In addition,the effect of pGPU6-sh4 is the best.3.Results of immunofluorescence showed that,as to the stable transfected cells expressing overfull CDK4 and CDK4 mutant protein,the fluorescent cells were nearly100% and results of western blot showed the expression of GFP was significant.4.As to the knockdown about CDK4,results of western blot showed that CDK4 protein was successfully knockdown by pGPU6-sh1 and pGPU6-sh4.In addition,the effect of pGPU6-sh4 is the best.5.For the stable transfected cells,results of immunofluorescence showed that When CDK4 was overexpressed,p16 protein was up-regulated in the cytoplasm and down-regulated in the nucleus;when the mutant and the knockdown of CDK4 exists,p16 protein was down-regulated in the cytoplasm and up-regulated in the nucleus.Conclusion1.P16 protein was up-regulated in the cytoplasm but down-regulated simaltaneously in the nucleus when CDK4 was overexpressed.2.P16 protein was down-regulated in the cytoplasm but up-regulated in the nucleus simaltaneously when CDK4 was mutated in its R24 site.3.P16 protein was down-regulated in the cytoplasm but up-regulated in the nucleus simaltaneously when endogenous CDK4 was knocked down.