Effect of NOR1 gene knockdown on the biological behavior of HeLa cells.
10.3969/j.issn.1672-7347.2014.08.001
- Author:
Yixin TAN
1
;
Wenjuan LI
;
Mei YI
;
Wei WANG
;
Pan ZHENG
;
Haijing ZHANG
;
Bo XIANG
;
Guiyuan LI
Author Information
1. Department of Dermatology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
- Publication Type:Journal Article
- MeSH:
Apoptosis;
Caspase 9;
metabolism;
Cell Survival;
Down-Regulation;
Gene Knockdown Techniques;
Genetic Vectors;
HeLa Cells;
Humans;
Hydrogen Peroxide;
Membrane Transport Proteins;
genetics;
metabolism;
Proto-Oncogene Proteins c-bcl-2;
metabolism;
RNA, Messenger;
Transfection;
Up-Regulation
- From:
Journal of Central South University(Medical Sciences)
2014;39(8):757-763
- CountryChina
- Language:Chinese
-
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.
