Purpose: Introducing the effect of
RNAi in fungi to downregulate essential genes has made it a powerful tool to investigate
gene function, with potential strategies for novel
disease treatments. Thus, this study is an endeavor to delve into the
silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant Aspergillus
flavus as the target genes.
Methods: In this
study, we designed three cyp51A-specific siRNAs and three MDR1-specific siRNAs
and after the co-transfection of siRNA into Aspergillus flavus, using
lipofectamine, we investigated the effect of different siRNA concentrations (5,
15, 25, 50nM) on cyp51A and MDR1 expressions by qRT-PCR. Finally, the Minimum Inhibitory Concentrations (MICs) of
voriconazole for isolates were determined by broth dilution method.
Results: Cyp51A siRNA induced 9, 22,
33, 40-fold reductions in cyp51A mRNA expression
in a voriconazole-resistant
strain following the treatment of the
cells with concentrations of 5, 15, 25, 50nM siRNA, respectively. Identically,
the same procedure was applied to MDR1, even though it induced 2, 3, 4, 10-fold
reductions. The results demonstrated a MIC for voriconazole in the untreated
group (4µg per ml), when compared to the group treated with cyp51A-specific
siRNA and MDR1-specific siRNA, both at concentrations of 25 and 50nM, yielding
2µg per ml and 1µg per ml when 25 nM was applied and 2µg per ml and 0.5µg per
ml when the concentration doubled to 50 nM.
Conclusion: In this study, we
suggested that siRNA-mediated specific inhibition of cyp51A and MDR1 genes play
roles in voriconazole-resistant A.flavus strain and these could be apt
target genes for inactivation. The current study promises a bright prospect for
the treatment of invasive aspergillosis through the effective deployment of
RNAi and gene therapy.