[Recombinant lentivirus-mediated gene transfer of NT4-p53(N15)-Ant inhibits the growth of hepatocellular carcinoma cells in vitro].
OBJECTIVE
To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy.


METHODS
The gene of p53(N15)-Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53(N15)-Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53(N15)-Ant was subcloned into the plasmid of lentivirus and cotransferred into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of LV. NT4-p53(N15)-Ant on HepG2 cells was measured by a colorimetric 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibition effect on HepG2 cells of LV. NT4-p53(N15)-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTT, LDH-release assay and annexin V-PI double staining.


RESULTS
The gene of p53(N15)-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines (1 x 10(11) pfu/ml), and the expression of NT4-p53(N15)-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4%, 46.9% and 33.9%, at 24 h, 48 h and 72 h, respectively, after infection by LV. NT4-p53(N15)-Ant. Compared with the LV. EGFP control group, there were significant differences (P < 0.01). The LDH level in HepG2 cells infected by LV. NT4-p53(N15)-Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV. EGFP group (P < 0.01), indicating the cell membrane destruction.


CONCLUSIONS
The recombinant lentivirus vector encoding gene NT4-p53(N15)-Ant is successfully constructed in this experiment by molecular cloning and recombination in vitro techniques, and the results suggested that this fusion gene has an anti-tumor effect, which provides the basis for further research on recombinant adenovirus for cancer gene therapy.
Song
Li-ping
LP
Department of Radiation Therapy, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an 710061, China. xaslp@126.com
Li
Yue-ping
YP
Qiu
Shu-dong
SD
Wang
Ning
N
D002470:Cell Survival; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D033704:Nucleotide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D009414:Nerve Growth Factors; D033704:Nucleotide Transport Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D007770:L-Lactate Dehydrogenase; C073922:neurotrophin 4