HIF-1α RNA I Sensitizes SPCA-1 Cells To Radiation

Objective:About 80% of all lung cancer cases are NSCLC, there is a difficult in increasing the effect. Hypoxia is one of the reasons, during this process, hypoxia-induced factor-1 plays a key role. The object of this study is to investigate the probability of RNA-interference targeting HIF-1α on the lung adenocarcinoma cells (SPCA -1) in radiosensitivity and to determine the radiation sensitivity of RNA interference for HIF-1α on the transplant tumor using polycationic polyethyleninine ( PEI ) , as a new kind pf gene vector.Method and materials:1. According to HIF-1α in GenBank data base, we designed the sense and antisense of RNAi.2.We insert the siRNA into the vector psuper at the restriction nuclease sites Bgl Ⅱ and Hind Ⅲ orientately, and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing.3.We transfected SPCA-1 cell with pSUPER-HIF-1α by lipofectamine 2000.4. The expression of protein was examined by Western blot.5. The expression of mRNA is examined by realtime RT-PCR under hypoxia and normoxia condition.6. The radiosensitivity of SPCA-1 cell of RNA-interference targeting HIF-1α was evaluated by constructing the cell survival curve.7. To establish the SPCA-1 nude mouse model;8. nude mice bearing SPCA-1 were divided into 3 treated groups and one control group.9. The expression of HIF-la was studied by immunohistochemical method after RNA interference for HIF-la. The difference of the volume, weight, survival time of the transplant tumor was studied among the simple radiation group, the simple RNA interference for HIF-la group and the combination of radiation and RNA interference for HIF-1.Results:l.we have inserted the HIF-la DNA to the vector psuper at the restriction nuclease site Bgl II and Hindlll orientately and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing.2. HIF-la protein level was decreased as the expression of Western blot.3. HIF-la mRNA was decreased as the expression of realtime RT-PCR under the condition of hypoxia and normoxia4. The radiosensitivity of SPCA-1 cell was increased by RNA-interference targeting HIF-la, the sensitivity enhance ratio is 1.5.5. The expression of HIF-la was decreased. RNA interference for HIF-1 combined radiation decreased the volume, weight and prolonged survival time of the transplant tumor significantly than other methods.Conclusion:1. RNA-interference targeting HIF-la may increase the radiosensitivity of SPCA-1 cell under hypoxia.2. In vitro, RNA-interference targeting HIF-la may increased the radiosensitivity of the transplant tumor using PEI as a new kind of gene vector.