Effect Of Silencing HIF-1α By RNA Interference On Biological Behavior Of Interference On Biological Benavior Of Acute Promyelocytic Cell Line NB4

Objective:To investigate the effects of RNA interference targeting HIF-1α on biological behavior of the proliferation, invasion and differentiation in NB4 in hypoxia condition, and to explore the mechanism of HIF-1α in the differentiation of acute myeloid leukemia cells.Methods:HIF-1α specific siRNA were designed and synthesized, recombinant lentiviral of HIF-1α-siRNA and negative control group were constructed and we screened out the best slow virus vector by transfection. In the CoCl2 simulated hypoxia microenvironment, the effect of different concentration gradient on the proliferation of NB4 cells was detected, and the concentration of CoCl2 which is the most favorable for the growth of NB4 cells was selected as the final concentration of simulated anoxic microenvironment. The expression of HIF-la was interfered with the best lentivirus RNAi vector in NB4 cells, mRNA expression level of HIF-la was tested by RT-PCR and the interference effect of protein including HIF-la, VEGF and SDF-1 were detected by Western blot. Cell survival rates of the test group and control group were detected with new tetrazole monosodium salt Kit (CCK-8), the changes in apoptosis level of the experimental group and control group under the interference of HIF-la were tested with annexin V-PE apoptosis detection reagent kit, the CDllb expressions of the experimental group and control group were detected by flow cytometry to analyse NB4 mature degree of differentiation and explore the mechanism of HIF-la in acute promyelocytic myeloid leukemia cell differentiation.Results:1. In the simulated hypoxia microenvironment, it had the strongest effect on NB4 cells when the final concentration of CoCl2 was 100μmol/L, the proliferation efficiency and RNA expression of HIF-la were significantly higher than the normal oxygen condition (P< 0.05).2. When MOI was 25,50,75, the average rate of GFP expression was 50.6%,75.3%, 98.2%, there were significant differences between the three groups (P< 0.01), so the choice of MOI for 75 is a comparatively reasonable selection.3. GFP expression intensity of transfected cells were tested by flow cytometry to assess transfection efficiency of the lentivirus interfering cell line NB4, and compared with negative slow virus, slow virus 4 for fluorescence transfection efficiency of NB4 cells is the lowest (P< 0.01), slow virus 1, slow virus 2, slow virus 3 transfection efficiency is high with no significant difference (P> 0.05); the expression of HIF-1αRNA of transfected cells was detected by RT-PCR, and compared with negative control, the HIF-la expression rate of slow virus 2 transfected NB4 cells was the lowest (P< 0.01) and slow virus vector 2 interfered NB4 cells can be used as experimental group.4. Compared with the negative control group, the rate of proliferation of cells and the expression of HIF-1α mRNA inhibition rate in the experimental group had significant difference (P< 0.01); the gray of HIF-la protein, VEGF and SDF-1 protein (P< 0.05); the apoptosis rate, cell invasion, cell differentiation and migration of experimental group had significant difference (P< 0.01). Cell morphology showed that the number of cells in the experimental group increased significantly (P< 0.01).Conclusions:Through the study found that HIF-la gene regulating HIF-la, VEGF and SDF-1 protein to accommodate the cell biological activity, and plays an important role on NB4 cells proliferation; RNA interference targeting HIF-la gene, in hypoxia condition, the expression of HIF-la RNA genes and protein including HIF-1α,VEGF and SDF-1 were all down regulated, experimental cell proliferation was inhibited, cell apoptosis rate increased greatly, differentiation and maturation ratio increased significantly. At the same time, we found that CD11b can be used as the detective index of early maturity differentiation of leukemia cells. This study using RNA interference, downregulated the hypoxia induced signaling pathway target gene and protein expression to expand a new target gene for the treatment of acute promyelocytic leukemia (APL), evaluating the treatment effects of APL on the gene and protein level scientificly is a positive exploration of a new method of APL treatment.