Lentiviral vector-mediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma.
OBJECTIVE
iASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types. This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis.


METHODS
A pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The fragment containing the shRNA cassette was cloned to pLVCT-tTR-KRAB plasmid. The recombinant vectors were co-transfected with viral packaging mix into 293T cells, and viral supernatant was harvested to determine the titer. After treatment with or without doxycycline, HepG2 cells infected with virus were harvested and the expression of iASPP was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Its effects on tumor growth were characterized using MTS assay, soft agar colony formation, and flow cytometry analysis.


RESULTS
The lentiviral vector expressing shRNA that targets to the oncogene iASPP was constructed successfully. HepG2 infected with the lentivirus expressing shRNA against iASPP inhibited the expression of iASPP in the presence of doxycycline, which resulted in the repression of tumor cell proliferation and anchorage-independent growth potential.


CONCLUSIONS
The lentiviral vector-mediated tet-on system demonstrates efficient and inducible knockdown of iASPP in hepatocellular carcinoma cells. iASPP gene may be involved in tumorigenesis and progression of human tumors.
Pang
Ming-Shu
MS
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China.
Chen
Xia
X
Lu
Bin
B
Zhao
Jian
J
Li
Bo-Hua
BH
Wei
Yu-Quan
YQ
Guo
Ya-Jun
YJ
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; C119612:PPP1R13L protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012097:Repressor Proteins; D004318:Doxycycline