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.
Highlights
The molecular mechanisms underlying the invasion and metastasis of cancer cells have been a hot topic in cancer research (Declereck et al, 1992)
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 tissue inhibitor of matrix metalloproteinases (TIMPs)-2 was carried out
Correct orientation was verified by restriction endonuclease digestion
Summary
The molecular mechanisms underlying the invasion and metastasis of cancer cells have been a hot topic in cancer research (Declereck et al, 1992). Numerous studies have indicated that tissue inhibitor of matrix metalloproteinases (TIMPs) are involved in the regulation of extracellular. More attention has been paid to the transfection of TIMPs into cancer cells leading to increased expression of TIMPs, which may regulate the metabolism of extracellular matrix and affect invasion capability of cancer cells. A recombinant retroviral vector containing the human TIMP-2 gene was constructed and transfected into human cancer cell line (SGC-7901). In vitro construction of human full-length TIMP-2 gene was performed and, through. Cell electrophoresis, soft agar colony formation assay and in vitro invasion assay were performed to evaluate the effects of TIMP-2 transfection on the malignant phenotype of these cells
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