[Therapeutic effects of RNA interference targeting HIF-1 alpha gene on human osteosarcoma].
OBJECTIVE
To investigate the inhibition effect of the small hairpin RNA (shRNA) targeting HIF-1alpha gene on the growth of osteosarcoma in vitro and in vivo.


METHODS
The small hairpin RNA (shRNA) eukaryotic expression vector targeting HIF-1alpha gene, named pSilencer-HIF, was constructed and transfected into cultured human osteosarcoma cell of line SaOS-2 via liposome reagent. Then the osteosarcoma cells were cultured under chemical hypoxia conditions. The inhibition effects on HIF-1alpha gene were determined by semi-quantitative reverse transcription PCR and Western blot analysis. The in vitro cellular growth activities were assayed by MTT colorimetry. The cell apoptosis was studied by electron microscopy, TUNEL assay, and annexin V/PI double staining. Eighteen Balb/C mice were randomly divided into 3 equal groups to be inoculated with SaOS-2/shRNA, SaOS-2/neo (blank vector), or SaOS-2 subcutaneously respectively and then the appearance and size of tumors were observed. Four weeks later the mice were killed and the volumes of tumor were calculated so as to evaluate the therapeutic effects of shRNA.


RESULTS
The successful construction of pSilencer-HIF plasmid was identified with sequencing. After the shRNA expression vector was transfected into the SaOS-2 cells, the expression of HIF-1alpha gene was inhibited significantly (by 90%). The cellular growth activities in the SaOS-2 cells transfected with pSilencer-HIF plasmid decreased obviously in hypoxia culture. After 72 hours of exposure to hypoxia, electron microscopy and TUNEL assay showed classic apoptosis characters in the SaOS-2 cells transfected with pSilencer-HIF plasmid with an apoptosis rate of 18.71% +/- 0.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the nontransfected group (both P < 0.01). The growth speed and formation rate of xenograft tumor in pSilencer-HIF transfected mice slowed down significantly. A lot of necrotic tissues could be observed in the pSilencer-HIF transfected group by HE staining, however, there was no similar inhibitive effect in the control groups.


CONCLUSIONS
shRNA targeting HIF-1alpha gene blocks the hypoxia transduction pathway efficiently and inhibits the growth of osteosarcoma cells.
Wu
Qiang
Q
Department of Orthopaedic, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Yang
Shu-Hua
SH
Ye
Shu-Nan
SN
Wang
Rui-Ying
RY
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D012516:Osteosarcoma / Q000235:genetics / Q000628:therapy*; D034622:RNA Interference; D014162:Transfection