Effects Of Antisense Oligodeoxynucleotides Targeting Hypoxia Inducible Factor 1α On The Proliferation And Apoptosis In Glioma Cell Line U251

Object: hypoxia inducible factor 1 αwas blocked with antisense technology toexplore the effect of antisense oligonucleotides targeting HIF-1αon the proliferationand apoptosis in human glioma cell line U251, and to investigate the possibility andefficiency of gene therapy targeting HIF-1αon human glioma.Method: Chemical hypoxia was induced by incubation of U251 with cobalt chloride.HIF-1αAS-PS-ODN was transfected into U251 cell line mediated by Dosperliposomal transfection reagent.The transfection effects were confirmed byfluorescence microscopy. RT-PCR and immunocytochemistry were applied in thisstudy to detect and measure HIF-1αmRNA and protein expression in U251 cell.trypanblue exclusion method and MTT Cell Proliferation Assay were widely used toevaluate the inhibitory effect of proliferation(PI). Then apoptosis was observed byAO/EB　fluorescence microscopy, DNA agarose gel electrophoresis and terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Theexpression of P53,Caspase-3and Bcl-2 was detected by immunocytochemistry toexplore the possible mechanism of apoptosis induced by HIF-1αAS-PS-ODN.Cisplatin was also used with AS-PS-ODN to confirm the possible synergistic effect.Results: 1.Afetr HIF-1αAS-PS-ODN was transfected into U251cells, the expressionof mRNA and protein of HIF-1αdecreased obviously. 2. AS-PS-ODN at differentconcentrations could inhibit the growth of U251 cell line, the proliferation inhibitoryrate could be as high as(56.20±4.22)%,there were statistical differences betweenAS-PS-ODN group and any other control group(P<0.05). 3. U251 cells displayed thetypically morphological features of apoptosis by AO/EB　fluorescence microscopy.DNA ladder of agarose gel electrophoresis was the typically morphological feature ofapoptosis. TUNEL showed that the apoptosis index of AS-PS-ODN group could be ashigh as(32.00±2.50)%.There was statistical difference compared with other controlgroups( P <0.05). 4.The immunohistochemical staining showed the number ofCaspase-3 positive cells increased singnificantly(P<0.05), that of Bcl-2 decreaced(P<0.05),while P53 changed negligibly( P>0.05). 5. AS-PS-ODN combination withcisplatin, the PI and AI reached(72.5±4.8)% and(42.0±3.5)%respectively. Therewas statistical difference compared with other control groups(P<0.05).Conclusion: 1. AS-ODN can reduce HIF-1αexpression in U251 cells significantly atlevels of protein and mRNA and it can also effectively suppress proliferation andinduce apoptosis. 2 Antisense inhibition of hypoxia inducible factor 1αcan induceP53-independent apoptosis. These results suggest that Bcl-2 and Caspase-3 maypossibly be involved in the regulation of apoptosis induced by treatment with HIF-1αAS-PS-ODNs in U251 cells. 3. HIF-1αASODN could enhance the sensitivity of U251cell to cisplatin. HIF-1αantisense treatment exerted an synergistic effect to thecytotoxicity of cisplatin.These results together indicate that suppression ofHIF-1αexpression may be a promising strategy that is selective for reducing thesurvival and facilitating chemotherapeutic efficacy of malignant glioma.