Objective: To construct hypoxic induction factor-1alpha (HIF-1alpha) siRNA expression cassette containing U6 promoter, alpha HIF-1alpha sense or antisense target sequence, and to observe its influence on the expression of cardiomyocytic HIF-1alpha during hypoxic state.
Methods: Neonatal murine cardiomyocytes cultured in the mixed gas were employed as the hypoxic model and were divided into normal control (cultured in normal oxygen), RNAi control (invalidated transfection interference sequence IV) and RNAi effective inhibition (effective transfection interference sequence, which was further divided into I, II and III groups according to the difference of downstream primer) groups. Three pairs (I, II and III) of PCR downstream primer containing HIF-1alpha encoded gene fragments (sense and antisense) and one pair of randomize sequence (IV) PCR downstream primer were designed and synthesized. U6 starter expression frame was constructed by PCR method. The cardiomyocytes were transfected simultaneously by sense and antisense sequence expression frame. Five plates of the cells were set at each time points in each group. The expression of HIF-1alpha mRNA was detected by RT-PCR at 6 hours of hypoxia. The change in the protein expression level at 1 hour of hypoxia was determined by Western blot, and the interference effects were monitored by immunohistochemistry.
Results: The best inhibition fragment screened was group II sequence. After the transfection and hypoxic culture, it was found that the cardiomyocytic HIF1alpha mRNA and protein levels in RNAi effective inhibition group were evidently lower than those in normal control and RNAi control groups (P < 0.01). While the protein inhibition rate (60% - 80%) between the former group and normal and RNAi control groups was no difference (P > 0.05).
Conclusion: The expression of the HIF1alpha in hypoxic rat cardiomyocytes could be effectively inhibited by our constructed HIF1alpha siRNA expression cassette group II.