title: Effect of small interfering RNA targeting hypoxia-inducible factor-1α on radiosensitivity of PC3 cell line.
abstract: OBJECTIVE
  To evaluate the effect of silencing hypoxia-inducible factor-1α (HIF-1α) expression by small interfering RNA (siRNA) on the radiosensitivity of the PC3 cell line.
  
  
  METHODS
  The expression of HIF-1α in PC3, a p53-null and androgen-independent prostate cancer cell line, was knocked down by siRNA. Irradiation was performed at 48 hours after transfection. The cells were divided into 3 groups: the PC3 group, control group (transfected with scramble siRNA), and HIF-1α silence group. HIF-1α expression was determined using real-time polymerase chain reaction and Western immunoblotting. A clonogenic assay and the cell counting kit-8 assay were performed to determine the radiosensitivity. Flow cytometry was used to assess apoptosis and cell cycle distribution.
  
  
  RESULTS
  HIF-1α siRNA downregulated HIF-1α expression in PC3 cells on the mRNA level and protein level, and its silencing effect on mRNA level was evident at 24-72 hours. The HIF-1α silence group had a low final slope of exponential part of a radiation survival curve, survival fraction of 2 Gy, quasi-threshold dose, and extrapolation number, and the sensitizing enhancement ratio was 1.24. The cell counting kit-8 assay showed decreased cellular viability (24 hours, F = 139.74, P < .01; 48 hours, F = 495.49, P < .01; 72 hours, F = 426.89, P < .01; 96 hours, F = 471.11, P < .01) in the HIF-1α silence group. Silencing HIF-1α also induced more apoptosis (PC3, 17.9% ± 1.65%; control group, 18.6% ± 1.37%; HIF-1α silence group, 29.1% ± 2.16%; F = 169.9, P < .01) and cell cycle arrest at the S, G(2)/M phase.
  
  
  CONCLUSIONS
  The suppression of HIF-1α in PC3 cells sensitizes the PC3 cells to irradiation. We have shown that HIF-1α inhibition attenuates repair of postradiation injury, with an increase in both interphase death and reproductive death after irradiation, apoptotic potential, and cell cycle arrest at the proliferative phase.
authors.affiliation: Department of Urology, First Affiliated Hospital of Soozhow University, Suzhou, Jiangsu, China.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Huang
authors.forename: Jiang
authors.initials: J
authors.lastname: Yu
authors.forename: Chunyin
authors.initials: C
authors.lastname: Yan
authors.forename: Jianquan
authors.initials: J
authors.lastname: Hou
authors.forename: Jingxian
authors.initials: J
authors.lastname: Pu
authors.forename: Guangbo
authors.initials: G
authors.lastname: Zhang
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Fu
authors.forename: Xizhi
authors.initials: X
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D015153:Blotting, Western; D059447:Cell Cycle Checkpoints / Q000235:genetics / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D015536:Down-Regulation / Q000502:physiology; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D011471:Prostatic Neoplasms; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011836:Radiation Tolerance / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D014410:Tumor Stem Cell Assay
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering