ABSTRACT
OBJECTIVE: To observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells. METHODS: The eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay. RESULTS: SHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05). CONCLUSION: SHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.
Subject(s)
Breast Neoplasms/pathology , Cell Proliferation , Gene Transfer Techniques , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Breast Neoplasms/genetics , Cell Line, Tumor , Female , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolismABSTRACT
OBJECTIVE: To construct a retrovirus-mediated expression system carrying human SHP-1 gene to transfer SHP-1 gene in human breast cancer MDA-MB-231 cells. METHODS: The full-length SHP-1 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line MCF-7 over-expressing SHP-1 protein. The gene fragment was inserted into the vector pLNCX2 to construct the recombinant retroviral plasmid, which was transfected into the packaging cell PT67 via Lipofectamine2000. A cell line stably producing the virus was selected with G418. MDA-MB-231 cells was infected with the virus, and the expression of SHP-1 gene in the positive cell clone was detected with Western blotting. RESULTS: A 1.8 kb cDNA fragment of SHP-1 gene was obtained from MCF-7 cells and successfully inserted into the pLNCX2. A stable cell clone PT67/SHP-1 and virus supernatant were obtained. Expression of SHP-1 protein was detected in the cells infected with the virus. CONCLUSION: The recombinant retroviral vector carrying SHP-1 gene has been successfully constructed and MDA- MB-231/SHP-1 cell line expressing SHP-1 has been obtained to allow further functional study of SHP-1 in breast cancer.
