Effect of NOR1 gene knockdown on the biological behavior of HeLa cells / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences)
;
(12): 757-763, 2014.
Article
in Chinese
| WPRIM
| ID: wpr-815531
ABSTRACT
OBJECTIVE@#To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma.@*METHODS@#The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot.@*RESULTS@#NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased.@*CONCLUSION@#Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Membrane Transport Proteins
/
RNA, Messenger
/
HeLa Cells
/
Transfection
/
Down-Regulation
/
Up-Regulation
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Cell Survival
/
Apoptosis
/
Proto-Oncogene Proteins c-bcl-2
/
Caspase 9
Limits:
Humans
Language:
Chinese
Journal:
Journal of Central South University(Medical Sciences)
Year:
2014
Type:
Article
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