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Published on: February 2, 2018
Lentiviral vector-mediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma
Ming-Shu Pang1, Xia Chen, Bin Lu
1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China.
Background And 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.
Insights
We developed a doxycycline-inducible lentiviral system to reduce inhibitory apoptosis-stimulating protein of p53 (iASPP) in liver cancer cells. This knockdown suppressed tumor cell growth and proliferation, suggesting iASPP’s role in tumorigenesis.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Inhibitory apoptosis-stimulating protein of p53 (iASPP) is upregulated in various human cancers.
- iASPP plays a role in inhibiting apoptosis and promoting cell survival.
Purpose of the Study:
- To create an efficient, doxycycline-regulated lentiviral system for inducible knockdown of iASPP.
- To investigate the preliminary functional role of iASPP in hepatocellular carcinoma (HCC) cells.
Main Methods:
- Construction of lentiviral vectors expressing shRNA targeting iASPP.
- Infection of HepG2 cells with lentivirus and induction with doxycycline.
- Analysis of iASPP expression via RT-PCR and Western blot.
- Assessment of tumor cell proliferation, anchorage-independent growth, and apoptosis using MTS assay, soft agar assay, and flow cytometry.
Main Results:
- Successful construction of lentiviral vectors for iASPP knockdown.
- Doxycycline-inducible and efficient downregulation of iASPP expression in HepG2 cells.
- Significant repression of tumor cell proliferation and anchorage-independent growth upon iASPP knockdown.
Conclusions:
- The developed tet-on lentiviral system provides effective, inducible knockdown of iASPP in HCC cells.
- iASPP is implicated in the tumorigenesis and progression of human cancers.
- Targeting iASPP may represent a potential therapeutic strategy for cancer treatment.
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