Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.
BACKGROUND
This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.


METHODS
ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG2-ANGPTL4 cells in vivo and in vitro, respectively.


RESULTS
The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 x 10(6) infective viral grains/ml, and the rate of HepG2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG2-ANGPTL4 cells than in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was higher than in HepG2-pMSCV cells (154% higher) or HepG2 cells (161% higher). The proliferation rate of HepG2-ANGPTL4 cells in vitro was obviously lower than those of HepG2-pMSCV cells and HepG2 cells (P <0.01). The mean volume and weight of tumors seeded from HepG2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG2 cells and HepG2-pMSCV cells (P <0.01).


CONCLUSIONS
A stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.
Li
Ke-qiang
KQ
Department of General Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China. lkq222jxmc@163.com
Li
Wen-lin
WL
Peng
Shu-you
SY
Shi
Xiao-yu
XY
Tang
Hong-lin
HL
Liu
Ying-bin
YB
D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins; D000818:Animals; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008113:Liver Neoplasms / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C474772:ANGPTL4 protein, human; D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger