Relationship between cathepsin L and invasion and metastasis of ovarian carcinoma cells

Abstract

To clone cathepsin L (CTSL) gene and construct the eukaryotic expression plasmid pcDNA3.1-CTSL and study the relationship between CTSL and invasion and metastasis in ovarian cancer cells in vitro. The total RNA was extracted from the ovarian cancer tissue and the intact cDNA of CTSL was applied by reverse transcription (RT)-PCR. The product of RT-PCR was cloned to pMD18-T vector, and subcloned to pcDNA3.1 vector. It was tested by the enzymation and DNA sequencing. The eukaryotic expression plasmid of CTSL was introduced into HO8910 cells by liposome transfection reagent. RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells. Western blot was used to confirm the CTSL protein expression in positive clones cells. The cell growth curves, clonogenicity efficiency were observed. The cell cycles were measured by flow cytometer. The ability of invasion, metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay, transwell migration assay and adhesion assay, respectively. The results from restrictive enzyme analysis and sequencing showed that the CTSL gene was successfully inserted into pcDNA3.1. Result from RT-PCR and western blot showed that the ovarian cancer cells which transfected by recombinant plasmid could express CTSL gene and protein. There was no difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in proliferation and adhesion ability (0.16 ± 0.04 versus 0.19 ± 0.04) of the cells (P > 0.05). There was difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in matrigel invasion ability (0.34 ± 0.18 versus 0.17 ± 0.04) and metastasis ability (1.252 ± 0.114 versus 0.486 ± 0.027) of cancer (all P < 0.05). CTSL maybe increase the ability of invasion and metastasis of ovarian cancer cells in vitro, which may be a molecular target of blocking invasion and metastasis of ovarian cancer.