An In Vitro Study: The Effect Of Cystatin M And Cathepsin B Single/Double Gene Coexpression Vector On Invasiveness And Metastasis Of Human Breast Cancer Cell Line

Objective To investigate the effect of Cystatin M and antisense Cathepsin B expression on malignant properties of human breast carcinoma MDA-MB-231 cells, CST6 and antisense CB single/double gene coexpression vectors were constructed and transfected into MDA-MB-231 cells.Cells proliferation,adhesion,migration and invasion of the extracellular matrix were evaluated in vitro.Methods Human CB gene fragment from placenta was amplified by one-step RT-PCR,and oppositely inserted into pBudCE4.1 and pBudCE4.1-CST6 respectively. The positive recombinants were identified by PCR,restriction endonuclease digestion and DNA sequence analysis.After successful construction of pBudCE4.1-CB and pBudCE4.1-CB-CST6,the recombinants were transfected into MDA-MB-231 cells,a highly invasive human breast cancer cell line by lipofectin technique.The expressions of CST6 and CB were detected by Western-blot.The effects of CST6 and antisense CB on the proliferation of MDA-MB-231 cells were detected by MTT assays and immunohistochemistry(IHC).With MTT assays,the adhesive ability to matrigel and FN was measured.The effects of CST6 and antisense CB on migration and invasion of the transfected cells were examined by reconstituted matrigel invasion and polycarbonate filters migration experiments.Results The recombinant vectors pBudCE4.1-CB and pBudCE4.1-CB-CST6 were constructed.They were transiently transfected into MDA-MB-231 cells.As compared with the parental and empty vector controls,the cells transfected with the single (pBudCE4.1-CB/pBudCE4.1-CST6) constructs revealed a marked reduction in tumor growth,adhesion,migration and invasion(P<0.01).And the effects with coexpression vector(pBudCE4.1-CB-CST6) were higher than the single constructs(P＜0.01).Conclusion Human CST6 and antisense CB eukaryotic single/double gene coexpression vectors were constructed for the first time.Our results indicated that CST6 and antisense CB single/double gene coexpression vectors play significant roles in inhibiting proliferation,adhesion,migration and invasion of MDA-MB-231 cells in vitro.Hence the recombinant coexpression vector may be a potential vector for suppressing metastasis of breast cancer and will be helpful in gene therapy for human breast cancer.