Hepsin蛋白對人類癌細胞的影響
The Effect of Hepsin Expression in Human Cancer Cell Lines
Date Issued
2004
Date
2004
Author(s)
Wang, Chung-Lin
DOI
zh-TW
Abstract
Hepsin is a type II transmembrane serine protease, which is present in most tissues, with the highest level expressed in liver. Previous studies suggest that hepsin is involved in cell growth and migration. It is also shown to interact with coagulation Factor VII, and convert zymogen factor VII to factor VIIa. However, embryonic developmental defects and hemorrhage abnormality were not shown in hepsin knockout mice. Hence the biological role of hepsin remains unclear.
To dissect the biological function of hepsin, we tried to express hepsin in cancer cell lines. After screening 4 cell lines with RT-PCR, we found that HeLa and Sk-Hep1 cells do not express hepsin. These two cell lines were transfected with Flag-Tetracysteine (TC) tagged-Hepsin expression vector. Besides wild type hepsin, we prepared 3 mutated hepsin expression vectors: activation domain mutation in residue R162A、S353Y、R162AS353Y ; Residue R162 is in activation domain and S353 is in enzymatic functional domain. Flag-TC tagged hepsin fusion protein can be detected with anti-Flag antibody with immunofluorescence staining and FlAsH-EDT2 staining. After G418 selection, we can only get cell lines with stably expressed mutated hepsin in both HeLa and Sk-Hep1 cells but not those with wild type hepsin fusion proteins. During transient expression, wild type hepsin fusion protein can be detected, but it was not able to cleave FVII to FVIIa in vitro. To prove the hepsin is involved in tumor cell growth in vivo ,we transplanted the wild type and mutant type hepsin-expressed HeLa cells via subcutaneous injection to nude mice. The growth rate and tumor size of hepsin-expressed HeLa cells in nude mice are significantly different. It suggests that hepsin may be involved in the regulation of cancer cell growth.
Subjects
第二型穿膜絲胺酸蛋白酶
裸鼠
人類癌細胞
細胞活體標定
hepsin
serine protease
flag tagging
tetracystei
SDGs
Type
other