Effect Of HIF-1α On Biological Characteristics Of Uterine Cervix Cancer Cell SiHa

Objective:To explore the effect of HIF-1αon biological characteristics of uterine cervix cancer cell SiHa and elucidate the related mechanism.Methods:1. Full length HIF-1αgene was cleaved from pSPT18-full length HIF-1αand ligated into pcDNA3.1. The reconstructed plasmids were transformed into competent bacteria and selected with antibiotics. The positive clones were conformed by restrictive enzyme digestion and DNA sequencing.2. Uterine cervix cancer SiHa cells were transfected with pcDNA3.1-full length HIF-1αand pcDNA3.1-dominant negative HIF-1αplasmids through lipofectamine. Transfectants were selected with G418 at 300mg/mL and cloned by a limiting dilution method. Theywere maintained in the presence of 150mg/mL of G418.3. The expression of HIF-1αand VEGF proteins were detected by immunocytochemical method and/or Western blotting.4. Detection of cell characteristics: cells were cultured in the medium contained different-concentrations of COCl₂ (0, 50μmol/L, 100μmol/L, 150μmol/L, 200μmol/L). The morphologic changes were observed continually by light microscope, the growth proliferation of cells was surveyed by the MTT assay, and cell apoptosis was detected through TUNEL assay.Resluts:1. Eukaryotic expression vector pcDNA3.1-full length HIF-1α(fL HIF-1α) was constructed successfully and confirmed by restriction endonuclease digestion and DNA sequencing.2. Immunocytochemical analysis showed that the integrated optical density (IODs) of HIF-1αprotein in fL HIF-1αgroup, dn HIF-1αgroup, pcDNA3.1 group and untransfected group were 97728.298, 3055.799, 4295.064, 4543.741 respectively. There was a significant difference between fL HIF-1αgroup and the others three groups (P＜0.05). There's no obvious difference among dn HIF-1 group, pcDNA3.1 group and untransfected group (P＞0.05). The IODs of VEGF protein in fL HIF-1αgroup, dn -HIF-1αgroup, pcDNA3.1 group and untransfected group were 12945.65, 6581.310, 6638.807, 7183.682 respectively. There was a significant difference between fL HIF-1αgroup and the others three groups (P＜0.05), and the difference between dn HIF-1 group and the other three groups was evidently significant (P＜0.05).3. Western blotting showed that the relative level of HIF-1αin fL HIF-1αgroup, dn HIF-1αgroup, pcDNA3.1 group and untransfected group was 1.29, 0.72, 0.77, 0.79 respectively. There was a significant difference between fL HIF-1αgroup and the others three groups (P＜0.05). There's no obvious difference among dn HIF-1 group, pcDNA3.1 group, and untransfected group (P＞0.05). The relative level ofVEGF in fL HIF-1αgroup, dn HIF-1αgroup, pcDNA3.1 group and untransfected group was 2.09, 1.05, 1.35, 1.44 respectively. There was a significant difference between fL HIF-1αgroup and the others three groups (P＜0.05). The difference between dn HIF-1 group and the other three groups was evidently significant (P＜0.05). There's no obvious difference between the pcDNA3.1 group and untransfected group (P＞0.05).4. MTT assay showed that the proliferation ability of fL HIF-1 group was obviously higher than that of dn HIF-1αgroup, pcDNA3.1 group and untransfected group (P＜0.05) no matter in the condition of normal oxygen or in the condition of chemical hypoxia by COCl₂. On the contrary, the proliferation ability of dn HIF-1αgroup was lower than that of the other three groups (P＜0.05). There was no significant difference between pcDNA3.1 group and untransfected group (P＞0.05).5. All cells grew well in the condition of normal oxygen. They all underwent apoptosis after treated with COCl₂ (200μmol/L) for 24 hours, the characteristic apoptotic morphology could be observed such as cell shrinking, blowing up, karyopyknosis, shedding and increased cellular fragments. There were more cells with dividing phase and less cells underwent apoptosis in fL HIF-1αgroup than in the other groups.6. TUNEL assay showed that the apoptosis rates of fL HIF-1αgroup, dn HIF-1αgroup, pcDNA3.1 group and untransfected group were displayed 22.52%, 66.86%, 36.12%, 38.0% respectively. There was a significant difference between fL HIF-1αgroup and the others three groups (P＜0.05). The difference between dn HIF-1αgroup and the other three groups is evidently significant (P＜0.05). There's no obvious difference between the pcDNA3.1 group and untransfected group (P＞0.05).Conclusion:1. The sequences of dominant negative HIF-1α(dn HIF-1α) and full length HIF-1α(fL HIF-1α) were in accordance with the sequences of genebank (AF208487). Cell clones which stably express dominant negative HIF-1α(dn HIF-1α) and full length HIF-1α(fL HIF-1α) were constructed. 2. Full length HIF-1αpromotes the proliferation of uterine cervix cancer cell SiHa and inhibits its apoptosis induced by COCl₂ chemical hypoxia. However, dominant negative HIF-1αevidently inhibits the cell proliferation and enhance cell apoptosis, suggest dominant negative HIF-1αhas potential usage to treat cancer.