Construction of retroviral recombinant containing human tissue inhibitor of metalloproteinase-2 (TIMP-2) gene and spontaneous invasion of gastric carcinoma cell lines <I>in vitro</I>

Abstract

Recombinant retroviral vector containing human tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene was constructed and investigation of the in vitro invasion and metastasis of gastric cancer cells transfected with TIMP-2 was carried out. Human TlMP-2 was isolated from recombinant vector Bluescript 1/TIMP-2(+), and then inserted into the retroviral vector pL-MT. Correct orientation was verified by restriction endonuclease digestion. Human full length TIMP-2 gene was ligated into a plasmid, which was then transfected into PA317 cell line. G418-resistant individual clones were selected to transfect human SGC-7901 cell line. Cell proliferation, cell electrophoresis, soft agar colony formation and in vitro
invasion were detected to analyze the bio-behavioral changes of cancer cells. The results from restriction endonuclease digestion were as theoretically expected. The cell electrophoresis rate, colony number and invasion ability in SGC-7901 cells and MFC cells transfected with TIMP-2 gene were significantly decreased when compared with control group. However, no significant changes were noted in the proliferation of cancer cells. We successfully construct a recombinant retroviral vector containing
human TIMP-2. TIMP-2 transfection could markedly alter the membrane charge of cancer cells, resulting in decreased electrophoresis capacity, cell migration and invasion. However, cell growth was not affected by TIMP-2. These results suggested TIMP-2 transfection might exert effects on the malignant phenotype of cancer cells through affecting extracellular environment, which provided a new way to investigate gene regulation of in vitro collagen metabolism.