Analysis Of CBP Gene Expression In Human Lung Cancer And Study On Depressant Effects Of CBP Eukaryotic Vector In SPC-A-1 In Vitro

CBP (csk-binding protein), a recently identified transmembrane protein, also known as phospoprotein associated with glycosphingolipid-enriched microdomains(PAG).The present studies demonstrate that CBP functions as a negative regulator of cell transformation and tumor cell growth through downregulation of Src activation and suppression of T-cell receptor activation. CBP might be broadly involved in SFKs (Src family tyrosine kinases) activated signaling pathways and tumorigenesis. Further study of CBP will supply a new way for molecular mechanisms of cancer.Objective: In this study, we analyzed expression of CBP mRNA and protein in primary human lung cancer cases by RT-PCR and Western-blot methods. The eukaryotic expression vector of CBP gene was constructed and transfected to human lung adenocarcinoma cell line SPC-A-1. Observed the biological behaviors of SPC-A-1 before and after transfection in vitro. All above studies has a purpose to explore the relationship between CBP gene and lung cancer and supply a new theoretical basis for genic therapy of lung cancer. Methods:(1) Semi-quantification expression analysis of CBP mRNA and protein in human lung cancer and corresponding normal lung tissues were performed by RT-PCR and Western-blot methods.(2)The total RNA was isolated from human lung cancer cell line SPC-A-1 and reversed into cDNA. Primers were designed according to human CBP mRNA from Genebank database and the target DNA fragments were amplified by RT-PCR. The eukaryotic expression vector was constructed by double restriction endonucleases cleavage directional clone method. (3)CBP eukaryotic expression vector was introduced into SPC-A-1 cells by liposome transfection reagent. Positive clones were screened with G418. PCR was used to confirm whether the recombinant vector DNA integrated with the genomic DNA of SPC-A-1 cells. Semi-quantification expression analysis of CBP mRNA and protein were performed in SPC-A-1 cells before and after transfection. (4) MTT growth test, wound-healing experiment, Matrigel invasion and movement assays were used in vitro to study the effect of CBP expression on transfected cells proliferation, movement and invasion. Results: (1)The mRNA and protein expression of CBP in human lung cancer tissues was significantly lower than that in corresponding normal lung tissues. (2)The construction of the eukaryotic vector was confirmed with plate screening, double endonucleases cleavage and sequencing identification. (3) PCR verified the successful integration of eukaryotic vector with the genomic DNA of SPC-A-1 cells. Semi-quantification RT-PCR and Western-blot showed that the CBP mRNA and protein expression was enhanced in transfected cells compared with that in untransfected cells. (4) Transfected with CBP eukaryotic expression vector could significantly inhibite growth, adhesion, invasion capabilities compared with the untransfected cells. Conclusions: (1)The mRNA and protein expression of CBP gene decreased significantly in human lung cancer tissue compared with that in corresponding normal lung tissue. (2)The eukaryotic expression vector encoding for human lung cancer CBP gene was constructed successfully.(3)The eukaryotic vector was transfected successfully to SPC-A-1 cells with liposome, which integrated with SPC-A-1 cell genome and markedly enhanced the expression of CBP mRNA and protein in SPC-A-1 cells.(4)The eukaryotic vector inhibite growth, adhesion, invasion capabilities of SPC-A-1 cell in vitro.