[Effect of silencing hypoxia-inducible factor-1 alpha by RNA interference on human breast carcinoma cell line].
OBJECTIVE
To investigate the effect of short hairpin RNA (shRNA) targeting hypoxia-inducible factor-1 alpha (HIF-1 alpha) on the human breast carcinoma MCF-7 cell line.


METHODS
The hypoxia environment was achieved by treating cells with cobalt chloride. The shRNA eukaryotic expression vector targeting HIF-1 alpha was constructed, and transfected into MCF-7 cells through lipofectamine 2000. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study the expression of vascular endothelial growth factor (VEGF). The mRNA and protein level of HIF-1 alpha were detected by real-time PCR and Western blot. Sub-G1 apoptotic population analysis, Annexin V/PI binding assay, and DNA ladder analysis were applied to investigate the cell apoptosis. The cell cycle was detected by flow cytometry.


RESULTS
The mRNA and protein level of HIF-1 alpha increased after exposure of MCF-7 cells to hypoxia (P < 0.01). However, apoptosis was lower in hypoxia compared with normoxia (P < 0.05). The HIF-1 level of MCF-7 transfected with HIF-1 alpha shRNA decreased approximately 91.63% (P < 0.01). When the cells were treated with or without apoptosis inducer Ara-C, the apoptosis of MCF-7 cells transfected with HIF-1 alpha shRNA increased by 1.75 times (P < 0.01) and 61. 31 times (P < 0.01), respectively. The expression of VEGF in MCF-7 cells transfected with HIF-1 alpha shRNA decreased 66.8% compared with untransfected cells (P < 0.05). Cell cycle progression was inhibited when the MCF-7 cells were transfected with HIF-1 alpha shRNA.


CONCLUSIONS
HIF-1 alpha plays an anti-apoptotic role in human breast carcinoma MCF-7 cell line. The shRNA we designed targeting HIF-1 alpha in MCF-7 can promote cell apoptosis, inhibit the expression of VEGF, and delay cell cycle progression.
Wang
Han
H
TEDA Life Science and Technology Research Center, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China.
Liu
Yong-jun
YJ
Han
Zhi-bo
ZB
Liang
Lin-hui
LH
Lv
Lu-lu
LL
Han
Zhong-chao
ZC
D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A