In Vivo Study Of Cxcl12-4EBP1D Gene Therapy In A Mouse Model Of Breast Cancer

Background:In human and animal breast cancers, the phosphorylations of eIF4E bing protein4EBP1are generally improved significantly, then causing increased availabilities of eIF4E and activities of the eIF4F complex, and selectively raise the expressions of some cell factors which can promote the cell proliferation, migration, angiogenesis, and resist the apoptosis, radiotherapy and chemotherapy, so as to promote the growth and vascularization of the tumor. Therefore, At present, the eIF4F complex has been seen as one of the key targets in the breast cancer therapy. Whether eIF4E binds to4EBP1or not is the key to the assembly of eIF4F translation initiation complex. The binding ability is regulated by the phosphorylation of4EBP1mediated by mTORCl. In the early stage of the study, we have found that the4EBP1mutant4EBP1D, whose RAIP motif and TOS motif are both deleted to avoid phosphorylation, can bind to the eIF4E, thereby blocks the eIF4F complex to assemble and inhibits the proliferation, migration of breast cancer cells and promote their apoptosis. However, it’s worth to notice that eIF4F complex plays important roles in the regulation of the body’s immune system and the function of hematopoietic system, so blocking the assembly of eIF4F complex may cause serious side effects. How to realize the specific inhibition to the eIF4F compounds of breast cancer cells is the focus of the contemporary study of tumor treatment.In recent years, studies have shown that the expression of CXCR4in breast tumor tissue increases obviously, and patients with breast cancers who have high CXCR4level have poor prognoses. At the same time, the excessive expression of CXCR4is closely related to cell proliferation, migration and invasion abilities of breast cancer cells. The chemokine CXCL12is the ligand of CXCR4, their combination activates the PI3K/Akt/mTOR pathway. If delete the55-68amino acids sequence of human CXCL12or make a proline mutation from glycine,it would loss the ability to activate the CXCR4, but can retain the binding ability. Objective:This study intends to target the CXCR4that is high expressed in breast cancer cells, and discuss the effect and safety of the phosphorylation defect type4EBP1D mediated by the the CXCL12mutant which has not the ability to activate the receptor CXCR4, in order to establish an effective and safe breast cancer gene therapy strategy for the experimental basis.Methods:1) Construct the eukaryotic expression vectors pSecX-4EBP1D, pCXCL12D and pCXCL12-4EBP1D which can respectively express protein transduction domain-4EBP1D mutant, CXCL12P2G mutant, CXCL12P2G mutant-4EBP1D mutant fusion protein by RT-PCR and molecular cloning technology. Then extract the plasmids using silica adsorption method and remove the endotoxin by isothermal Triton X-114method.2) To establish animal models of breast cancers in mice using4T1cells and transfect the empty carrier, pSecX-4EBP1D, pCXCL12D and pCXCL12-4EBP1D in vivo mediated by Invivojet PEI, in order to evaluate the effects of gene therapies.3) after the gene therapies, respectively collect the tumors, hearts, livers, lungs, kidneys from the CXCL12D treatment group, CXCL12-4EBP1D treatment group,4EBP1D treatment group and empty carrier control group to make the paraffin sections and HE staining, so as to judge the effects of gene therapies and the side effects to the important organs.Results:After19days of treatment, we found that compared with the empty carrier, the tumor growth in mice of the CXCL12D treatment group, CXCL12-4EBP1D treatment group,4EBP1D treatment group were all slow and the treatment effect of pCXCL12-4EBP1D was the most significant. HE staining showed that the liver and kidney in mice of4EBP1D treatment group appeared significant lesion features, which were not obvious in CXCL12D treatment group and CXCL12-4EBP1D treatment group.