[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.
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
Hai-yan
HY
Ye
Ai-lin
AL
Guo
Meng
M
Zhang
Guo-li
GL
Lü
Jian-ming
JM
Wen
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