[Construction of an eukaryotic expression vector for PRL-2 and its effect on human hepatocellular carcinoma cell invasiveness and migration in vitro].
OBJECTIVE
To construct an eukaryotic expression vector for PRL-2 and evaluate its effect on the invasiveness and migration of a human hepatocellular carcinoma cell line.


METHODS
RT-PCR was performed to amplify the complete PRL-2 open reading frame using the total mRNA of hepatocellular carcinoma HepG2 cells as the template. PRL-2 gene was inserted into the pGEM T easy vector and sequenced, and the correct PRL-2 sequence was subcloned into the mammalian expression vector pcDNA3.1+. The constructed PRL-2 vector was transfected into CL1 cells via lipofectamine, and the stable expression of PRL-2 mRNA was detected by RT-PCR, the expressed protein identified by immunohistochemistry and Western blotting, and the effect of PRL-2 on the adhesion ability of CL-1 cell evaluated with MTT assay 20 and 120 min after transfection. The effect of PRL-2 on the invasive migration of CL-2 cells was evaluated according to the number of cells penetrating the Matrigel layer of polycarbonate membrane of Boyden chamber.


RESULTS
RT-PCR yielded a fragment of 504 bp and the inserted PRL-2 sequence was verified by sequence analysis. The subclones were identified by restriction endonuclease digestion, and a G418-resistant clone, PRL-2-CL1, was obtained after 8 weeks of selection. RT-PCR showed stable expression of PRL-2 mRNA, and Western blotting confirmed overexpression of PRL-2 protein in the transfected cells. PRL-2 increased the adhesion rate of CL-1 cells to fibronectin at 20 min and 120 min after transfection (P<0.05), and also the number of CL-1 cells penetrating the polycarbonate membrane from 10.0+/-3.7 to 44.8+/-2.6 (P<0.05).


CONCLUSIONS
An eukaryotic expression vector of PRL-2 has been successfully constructed, which allows stable and efficient expression in CL-1 cell line. PRL-2 can promote cell adhesion and invasion activity of human hepatocellular carcinoma cells.
Ye
Hai-yan
HY
Department of Obstetrics and Gynecology, Center of Medical Research, People's Hospital of Guangdong Province, and Department of Pathology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. zhoudanyao@yahoo.com.cn
Guo
Ai-lin
AL
Zhang
Meng
M
Lü
Guo-li
GL
Wen
Jian-ming
JM
D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D002448:Cell Adhesion / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology*; D009361:Neoplasm Invasiveness / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C487561:PTP4A2 protein, human; D017027:Protein Tyrosine Phosphatases