[The overexpression and significance of MKP-1 in oxygen-deprived gastric cancer cell line SGC7901].
OBJECTIVE
To investigate the expression status of Mitogen-activated Protein Kinase Phosphatase-1 in oxygen-deprived gastric cancer cell line SGC7901 and its role in HIF-1 regulation.


METHODS
The expression of MKP-1 in gastric cell line SGC7901 was detected with Western blot and semiquantity RT-PCR; Eukaryotic sense expression vector was constructed based on DNA recombination technology. Transfections of SGC7901 were performed using liposome; The luciferase activity was determined using Dual Luciferase Reporter System and the levels of VEGF in SGC7901cells under normoxia and hypoxia were measured by ELISA.


RESULTS
(1) Semiquantity RT-PCR and Western blot suggested that the expression of MKP-1 was elevated in oxygen-deprived gastric cancer cell line SGC7901; (2) 48 hours after transfection, the phosphorylated form of HIF-1alpha in cell line transfected with recombinant plasmids was lower compared with that in cell line transfected with empty vectors after 12 hours of exposure to hypoxia; (3) There was very low luciferase activity under nomoxia while under hypoxia luciferase activity increased in a time-dependent manner and at all time points there was significant lower luciferase activity in SGC7901 cells than in cells transfected with empty plasmids. (4) At different points of time course, the expression of VEGF in SGC7901 was significantly higher under hypoxia than that in SGC7901 under nomorxia, while under hypoxia, the expression of VEGF in SGC7901 transfected with recombinant plasmids was significantly lower than that in SGC7901 transfected with empty vectors at all time points as indicated.


CONCLUSIONS
The expression of MKP-1 in SGC7901 was elevated under hypoxia, which could downregulate the HIF-1 trans-activition activity thereby repressing the expression of downstream target gene VEGF.
Liu
Chang-jiang
CJ
Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Shi
Yong-quan
YQ
Han
Zhe-yi
ZY
Xue
Yan
Y
Pan
Yang-lin
YL
Shen
Hui-qin
HQ
Du
Jing-ping
JP
Han
Shuang
S
Qiao
Tai-dong
TD
Fan
Dai-ming
DM
D015153:Blotting, Western; D018797:Cell Cycle Proteins; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054638:Dual Specificity Phosphatase 1; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000473:pathology; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases