Construct Eukaryotic Expression Vector Of Syk Gene And Study On Inhibiting Effect For Invasiveness Of Breast Cancer Cells In Vitro

Objective: Tumor threatens seriously the life and health of human. Breast cancer is a kind of malignant tumor with highest incidence in women. The disease incidence presents increasing tendency year by year. It is threatens women's health severely. Genesis and development of breast cancer are complicated processes with multi-procedure. It is correlated with expression of many genes and proteins closely, including proto-oncogene activation (such as HER2/neu), anti-oncogene mutation and loss (such as p53) and so on. Over the last several decades, gene alteration and function study in the process of breast cancer genesis, development and metastasis are always investigation focus in this field. Protein tyrosine kinase (PTK) is an zymoprotein that can catalyze substrate protein tyrosine residue phosphorylated. It is involved in many pathway of signal transduction, and plays an important role in controlling cell differentiation, proliferation and diffusion. Syk is a non-receptor type of protein tyrosine kinase. It is commonly expressed in normal mammary gland and epithelial tissue, and can promote the growth of human breast epithelial cells. It is discovered recently, Syk is an important suppressor factor in breast cancer cells'growth and metastasis. There is a low level of Syk expression in noninvasive breast cancer cell lines , and a lower or undetectable level in invasive breast cancer cell lines. Reduction or loss of Syk expression is involved with malignant tendency of human breast tissue, but its specific mechanism in inhibiting invasive growth of tumor cells is not clear. In this study, syk gene was clone into the expression vector of pcDNA3.1D/V5-His- TOPO, and then transfected into human breast cancer cells MDA-MB-231 by using lipofectamin protocols to observe the effect on cells induced by gene. It provides a foundation for further study on the mechanism of Syk to inhibit tumor cells'invasion and metastasis.Methods: 1 Construct syk gene eukaryotic expression vector Total RNA was isolated from human breast cancer cells MDA-MB-468, syk gene encoding fragment was amplified by RT-PCR and was cloned into the eukaryotic expression vector pcDNA3.1D/V5-His-TOPO. After PCR amplification, Apaâ… digestion and DNA sequencing confirmation, its eukaryotic expression vector pcDNA3.1D/V5-His-TOPO/syk was constructed successfully.2 Transfect into MDA-MB-231 cells Recombinant plasmid was transfected into human breast cancer cells MDA-MB-231 by using lipofectamin protocols. MDA-MB-231 cells do not express Syk. After G418 selection, MDA-MB-231/syk cells stable express Syk. The expression of Syk on transfected cells was analyzed by Western blot and RT-PCR methods.3 VEGF and MMP-9 expression analysis Using flow cytometry(FCM) and RT-PCR to detect the expression of VEGF and MMP-9 on three group cells:transfect syk cDNA group (MDA-MB-231/syk cell),transfect pcDNA3.1 group (MDA-MB-231/pcDNA3.1 cell) and none transfected group (MDA-MB-231 cell).4 Cell proliferation analysis Detect these cells'growth and reproductive activities by MTT and draw growth curve.5 Cell migration and invasion detection Using migration and invasion experiment to detect the cells'migratory and invasive abilities.Results: 1 Eukaryotic expression vector of pcDNA3.1D/ V5-His-TOPO/syk was constructed successfully.2 MDA-MB-231/syk cells stable express Syk.3 Compared with MDA-MB-231/pcDNA3.1 and MDA- MB-231 cells, MDA-MB-231/syk cells lower express VEGF and MMP-9.4 The growth and reproductive activities of MDA- MB-231/syk cells have no changed. But the migratory and invasive abilities have decreased.Conclusions: Potential tumor suppressor syk could transfect human breast cancer cells MDA-MB-231 by liposome efficiently. The migratory and invasive abilities of cells transfected syk gene reduced obviously. It has laid a solid foundation for further study of Syk suppressive effect in vivo and gene therapy in breast cancer.