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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
[Effect of NOR1 gene knockdown on the biological behavior of HeLa cells]
Yixin Tan1, Wenjuan Li, Mei Yi
1Department of Dermatology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
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.
Insights
Knocking down the NOR1 gene in cervical cancer cells enhances their viability and growth. This NOR1 gene silencing also reduces apoptosis, potentially by affecting Bcl-2 and caspase 9 pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Cervical carcinoma is a significant global health concern.
- The role of oxidored nitro domain containing protein 1 (NOR1) in cervical cancer progression is not well understood.
Purpose of the Study:
- To investigate the impact of NOR1 gene knockdown on the biological behavior of HeLa cells, a cervical carcinoma cell line.
- To elucidate the molecular mechanisms underlying NOR1's influence on cell viability, proliferation, and apoptosis.
Main Methods:
- NOR1 gene knockdown was achieved in HeLa cells using shRNA delivered via pSUPER.neo+GFP vector.
- Cell viability and proliferation were assessed using MTT assays.
- Apoptosis was evaluated using Hoechst 33258 staining and TUNEL assay following H₂O₂ treatment.
- Expression levels of NOR1, Bcl-2, cleaved caspase 9, and PARP were determined by RT-PCR and Western blotting.
Main Results:
- Successful construction of NOR1-knockdown HeLa cells was confirmed.
- NOR1 knockdown significantly increased HeLa cell viability and proliferation.
- Knockdown of NOR1 inhibited H₂O₂-induced apoptosis in HeLa cells.
- Western blot analysis revealed increased Bcl-2 expression and decreased cleaved caspase 9 and cleaved PARP levels in NOR1-knockdown cells.
Conclusions:
- Endogenous NOR1 silencing promotes cell viability and growth in HeLa cervical carcinoma cells.
- NOR1 knockdown attenuates H₂O₂-induced apoptosis, likely through the upregulation of Bcl-2 and downregulation of the cleaved caspase 9 pathway.
