Construction Of CXCL12-KDEL Fusion Gene And Inhibiting Metastasis Of Human Oral Squamous Cells By Capturing CXCR4 In Cell

SECTION ONE The relationship between expressions of CXCL12-CXCR4 and neck lymph node metastasis in head and neck squamous carcinomaObjective:Analyzing the relationship between the expressions of chemotatic factor CXCL12 and its acceptor CXCR4 in head and neck squamous carcinoma and clinical and pathological character of HNSCC,exploring the effect of CXCL12-CXCR4 biological axis on lymph node metastasis in HNSCC.Methods:The clinical,pathologic information of patients underwent surgical resection or biopsy with HNSCC from March2005 to January 2006 was collected and reviewed in department of head and neck,Tianjin cancer hospital.15 cases of normal mucosa and benign lesion were included as control.The different expressions of CXCL12-CXCR4 in different groups were investigated by immunohistochemical staining and RT-PCR to identify the relationship with lymph node metastasis.Results:Of the HNSCC's age,gender,tumor classification,cervical lymph node metastasis,the positive expressions of CXCR4 were 71.8%and 26.9%in stageⅢ-ⅣandⅠ-ⅡHNSCC,83.3%and 42.6%in Grade 3 and Grade 1/2 retrospectively.The differences were significant(P＜0.01).CXCR4 protein was expressed in 71.4%of HNSCC with lymph metastasis,in 33.3%without metastasis and in 13.3%of control group,the differences of them were significant(P＜0.05).Expressions of CXCR4 in control group were very low.CXCL12 expression was not significant different for all the parameters including lymph metastasis.Conclusion:Overexpression of CXCR4 was related to lymph node metastasis and classification in HNSCC.Expression of CXCL12 was significant higher in target organ(lymph node)than non-target organ(fatty tissue).CXCL12-CXCR4 biological axis might promote orientation metastasis to cervical lymph node in HNSCC. SECTION TWO Construction of pIRES2-EGFP eukaryotic vector carried CXCL12-KDEL fusion gene and blocking CXCL12-CXCR4 chemotaxis function in human high metastasis squamous cells by tranfection of CXCL12-KDEL geneObjective:To construct the pIRES2-EGFP eukaryotic vector carried CXCL12-KDEL fusion gene and inhibit the expression of CXCR4 of cell membrane and block chemotaxis of CXCL12-CXCR4 biological axis by tranfection of CXCL12-KDEL gene in oral high metastasis squamous cells.Methods:Searching the gene code of human CXCL12 in Genbank,we ascertained the area of amplification and designed the primer with the sequence of KDEL.The coding gene of human CXCL12-KDEL signal was cloned by RT-PCR from human cervical lymph node.We linked the pMD19-T vector to CXCL12-KDEL and subcloned CXCL12-KDEL fusion gene to eukaryotic vector pIRES2-EGFP.Oral high metastasis squamous cells were transfered of culture,and transfected using CXCL12-KDEL plasmid by liposome.The rate of transfection was observed.Cells which were transfected using the empty vector pIRES2-EGFP and normal cells were included as control groups.Function test were performed:CXCL12 protein marked with E-tag was examination by Western blot.Expression of CXCR4 of Tb3.1 cell membrane was detected by flow cytometry.Chemotaxis chamber were used to evaluate effect of various concentrations of CXCL12 on Tb3.1 cell migration.Results:DNA sequence analysis proved that the coding gene of human CXCL12-KDEL was cloned.We linked the pMD19-T vector to CXCL12-KDEL and constructed the CXCL12-KDEL- pIRES2-EGFP fusion gene successfully. CXCL12-KDEL gene was transfected in oral high metastasis squamous cells successfully.The rate of transfection was over 50%after 72h.Western blot showed the expression of E-tag(CXCL12)in transfected cells but not control groups and supernatants.FACS analysis showed that the expression of CXCR4 of membranes in experimental group(EG)was significant lower than two control groups(CG).The number of migrating cells in experimental group(EG)was significantly lower than that of control groups(CG)(P＜0.05).Conclusion:We constructed the CXCL12-KDEL- pIRES2-EGFP fusion gene successfully, which may be more helpful to further researches.By transfecting CXCL12 carrying an endoplasmic reticulum(ER)retention sequence into Tb3.1 cells,the receptor CXCR4 was retained in the ER and failed to reach the cell surface.The main role—chemotaxis of CXCL12-CXCR4 biological axis was blocked.