| Chemokine receptor 4 (CXCR4), as one of the seven-transmembrane G-protein-coupled receptor family, is a kind of cell membrane protein, whose chemokine that can bind and activate to which was only known to be Stromal cell-Derived Factor-1 (SDF-1). Due to the interaction between CXCR4 expressed highly on tumor cells and SDF-1 directly involving in the metastasis of tumor disease such as breast cancer, it maybe a new approach to effectively prevent the metastasis of breast cancer by blocking the functional expression of CXCR4 on the surface of tumor cell. In this study, on the basis relationship of CXCR4/SDF-1αstructure-activity relationship, vie an intrakine mutant strategy which contain SDF-1αmutant SDF-1α/54 gene and an endoplasmic reticulum (ER) retention signal (KDEL) to block the expression of CXCR4, the SDF-1α/54/KDEL chimeric gene were amplified and then subcloned into the eukaryotic expression vectors and the adenovirus vector respectively. The newly fusion protein SDF-1α/54/KDEL could be intracellularly expressed in the ER, and simultaneously could specifically bind to the newly-synthetized target receptor CXCR4 and consequently remain in ER, the compouds followed by the ubiquitin proteasome pathway or lysosomal pathway decomposition and so on, by which knockout the expression of CXCR4, thus inhibits cancer metastasis function phenotype. The results will also provide direct experimental evidence to explore CXCR4 as a target for the cancer gene therapy.Objectives:①The mature wild-type peptide-colding sequence (SDF-1wt) of hSDF was cloned from health adult myeloid tissue by RT-PCR, then its prokaryotic expression was constructed by orientally inserting into the prokaryotic expression plasmid pET-30a(+) . Furthermore, deletion mutation was applied by losing the structural domain of C extermial to form SDF-1α/54, which provided the substance for the practice of the intrakine mutant.②To construct the eukaryotic expression plasmid of SDF-1αmutant recombinant SDF-1α/54/KDEL, and investigate its effect on CXCR4 in T-cell leukemia cell line Molt-4 cells as well as the activity of SDF-1α/54/KDEL protein, all of which could provide feasibility for the knockout phenotype of tumor CXCR4 to inhibit tumor metastasis. ③To construct recombinant adenovirus expression vector Ad-SDF-1α/54/KDEL and inspecting their phenotype knockout activity such as the cell growth, chemokines and invasion for breast cancer cell line MCF-7.④To establish a proper model of human breast carcinoma with highly expressing CXCR4 in nude mice for preparation of the effects with purified Ad-SDF-1α/54/KDEL Methods:①Human SDF-1α/54 gene with the depletion ofα-helix at the C-terminal domain acquired by site-directed mutagenesis was obtained from wild-type hSDF-1wt gene with RT-PCR, and then hSDF-1wt and hSDF-1α/54 were inserted into the prokaryotic expression vector pET-30a(+) respectively and sequencely transform the E coli to express BL21(DE3) with the induction of IPTG..②SDF-1α/54 gene was amplified by PCR from SDF-WT- PET30a(+) plasmid, and the coding gene of KDEL corresponding to the rention signal sequence in ER was introduced into its C-terminal. SDF-1α/54/KDEL gene was subcloned into the eukaryotic expression plasmid of pcDNA3.1/Myc-His(-)A and pEGFP-C3 to construct two of expression plasmids: pcDNA3.1/SDF-1α/54/KDEL and pEGFP /SDF-1/54α/ KDEL. Exactly sequencing recons was used to transfect Cos-7 cell with liposome after plasmid isolation and purification, and the functional expression of SDF-1α/54/KDEL mutant protein was assayed by Western blot, as well as the location of fusion protein containing SDF-1/54α/KDEL in the plasmid transfected cell was observed by laser confocal microscopy. The recombinant vector of pcDNA3.1/SDF-1α/ 54/KDEL and pEGFP/SDF-1/54α/KDEL with large plasmids isolation abstraction and purification by electroporation were to transiently transfect CXCR4-positive Molt-4 human T-cell leukemic cell line, and the level of CXCR4 on cell surface was checked by FCM. The growth state of Molt-4 after transfection was examined by FCM, and the phaenotypic knockout activity of Molt-4 after transfected with SDF-1α/54/KDEL was proved by chemotaxis experiment.③SDF-1α/54/KDEL gene was amplifyed by PCR from pcDNA3.1/SDF-1α/54/ KDEL vector, and vacuous shuttle plasmid pAdTrack-CMV were cut by XbaI and XhoI respectively, following the connection of them. SDF-1α/54/KDEL was subcloned into the shuttle plasmid vector to construct pAdTrack-CMV-SDF-1α/54/KDEL, and then it was homologously recombinated with pAdEasy-1 by the means of insider homologous recombination of host bacterium to construct Ad-SDF-1α/54/KDEL. After exact assessment, Ad-SDF-1α/54/KDEL was intransfected in 293-cell mediated by Lipofectamine 2000, virus plaque appeared in 7 days after the transfection. The mRNA was extractedand then assayed by RT-PCR and SDF-1α/54/KDEL expression cluld be observed by GFP. Flourescence Recombinant AD virus was bulkly harvested by passages and the MOI value was assayed. The phenotypic knockout activity of Ad-SDF-1α/54/KDEL was exerted with in vitro. Observation Ad-SDF-1 /54/KDEL on breast cancer cell proliferation of MCF-7 by MTT methods; Ad-SDF-1α/54/KDEL on MCF-7 cell chemotactic activity; Gelatin zymography and invasion experimental verification the inhibitory effect of recombinant adenovirus Ad-SDF-1α/54/KDEL on MCF-7 cell.④The animal model of breast cancer metastasis was established by transplanting MDA-MB-231 cell into the subcutaneous fat pad of nude mice to detect the expression of SDF-1 and CXCR4 in the primary tumor tissue and metastatic tumor tissue in tumor-bearing mice, with an aim to get ready to investigate the metastasis role of Ad-SDF-1α/54/KDEL for the transfer of breast cancer.Results:①Human SDF-1 wild-type (SDF-1wt) gene was amplified by RT-PCR from adult bone marrow stromal cells; Using PCR to delete the SDF-1 C-terminalα-helix domain. The sequencing results showed SDF-1wt cloning and mutation is correct.②Enzymes digestion and sequencing results demonstrate successful construction of SDF-1α/54/KDEL gene recombinate eukaryotic vector: pcDNA3.1/Myc-His (-) A and pEGFP-C3. Western blot analysis confirmed transfected Cos-7 cells with the mutant SDF-1 /54/KDEL chimeric gene expression and further fusion protein intracellular localization. Furthermore, SDF-1α/54/KDEL and EGFP fusion protein was localized ER. Two recombinate eukaryotic expression vectors were transfected by electroporation to Molt-4 cell, this could significantly downregulation the expression of CXCR4. The positive rate of cells and fluorescence intensity are more than 90%, SDF-1α/KDEL and SDF-1α/54/KDEL effect have no significant difference. Molt-4 proliferation has not been affected after transient transfection detected by Flow cytometry (FCM); Chemotaxis experiments suggest that Molt-4 cells chemotactic role is inhibited.③The adenovirus shuttle vector pAdTrack-CMV-SDF-1α/54/KEL was constructed, and the recombinant adenovirus Ad-SDF-1α/54/KDEL was obtained by co-intransfecting pAdEasy-1 into 293 cells. RT-PCR and GFP expression identification showed there is the transcription and expression of SDF-1α/54/KDEL in the packaged virus. The plural MOI infection of amplified Adenoviruses was 0~200, and its infection efficiency for MCF-7 was 100% when MOI was 100. FCM analysis revealed that Ad-SDF-1α/54/KDEL infected within 10 days could still significantly down-regulate the expression of CXCR4 in MCF-7 cells. Cell growth and proliferation were not influenced to a certain extent of MOI 200. When MOI was 100, MCF-7 cells infected by Ad-SDF-1α/54/KDEL for 3 days can significantly inhibit its chemotaxis and invasion.④Nodules can be seen at the transplanting sites after implantting MDA-MB-231 breast cancer cells into nude mice BALB/C for seven days. The successful rate of tumor transplantation was 100% after the cancer cells were vivo domesticated and passaged for three generation. Pathology test results showed the positive expression of CXCR4 in the tissue of liver and lungs. RT-PCR of SDF-1αdisplayed it expressed in the tissue of spleen, liver, lung of nude mice, all of which contributed for the primary establishment of in vivo animal model for the Ad-SDF-1α/54/KDEL activity of breast cancer.Conclusions:①Human stromal cell derived factor-1 (hSDF-1) gene was successfully cloned, and the acquirement of SDF-1α/54 mutant could be done through deleting the C-terminalα-helix domain with the technique of gene deletion mutant, and so dose E. coli expression system..②SDF-1α/54/KDEL expressed in two different Eukaryotic expression vectors has the ability of CXCR4 phenotype knockout at gene-level after the transfection of Molt-4 cells by electroporation, and also has the ability to inhibit chemotaxis of Molt-4 cells and has no effect on the growth of Molt-4 cells. The activity of knockout phenotype were not affected by the loss of C-terminalα-helix domain of SDF-1α.The possible mechanism maybe that the post-transfected newly-synthesized SDF-1αprotein, due to the ER location function of KDEL, when it was transferred to Goergi, will not be transferred toward cell membrane and secreted out but remain in ER. Because the main structure of SDF-1α/54/KDEL with the ability to bind to CXCR4 has been reserved, it still has the ability to recognize and bind to CXCR4. Thus SDF-1α/54/KDEL protein could combine the newly synthesized or internalization CXCR4 to form a compound, then the compound may be breakdown by the pathway of ubiquitin proteolysis or other intracellular mechanism, resulting in the expression inhibition of CXCR4 on the cell membrane and the achievement of phenotypic knockout. ③Recombinate adenovirus Ad-SDF-1α/54/KDEL has a good activity of phenotypic knockout for breast cancer cells MCF-7, which was characteristics of time-prolonged action, high-titer, quick proliferation, high efficiency for action and low toxicity, thus make it to be an effective tool carrier for the breast cancer metastasis research. The inhibition function of CXCR4 knockout phenotype in vitro for breast cancer cell chemotaxis and invasion is based on the CXCR4 inhibition at the target.④A human breast carcinoma model in nude mice was established, which reserved certain biological features of CXCR4/SDF-1,therefore it may be an available model for the further research of the activity of Ad-SDF-1α/54/KDEL in vivo.
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