Abstract
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.
Highlights
In this study, we suggested that small-interfering RNAs (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
Effect of siRNA concentration on cyp51A and MDR1 expressions Cyp51A and MDR1 messenger RNAs (mRNAs) expressions were quantified by Quantitative Real-Time PCR (qRT-PCR) in a voriconazole-resistant A.flavus strain and a voriconazole-susceptible strain treated by the specific siRNA and positive control and compared with a negative control after 24h
Howard et al demonstrated that upregulation in cyp51A gene is associated with azole resistance in A.fumigatus,[32] but Liu et al indicated that the expression levels of cyp51A, cyp51B and cyp51C genes were not associated with voriconale-resistance in A. flavus,[8] whose results were in conflict with our findings
Summary
Aspergillus spores cause a broad-spectrum of diseases in humans, ranging from allergy-type illnesses to invasive infections depending on host immunity.[1,2] Since the last decade, invasive aspergillosis (IA) is associated with significant morbidity and mortality in hematological malignancies, bone marrow transplant (BMT) recipients and patients suffering from AIDS and chronic granulomatous diseases.[3,4] Of all the known Aspergillus species, approximately 80% of IA is caused by Aspergillus fumigatus; Aspergillus flavus is the second leading cause of IA in Western countries.[5,6] In certain climate and geographical locations like the Middle East, Africa and Southeast Asia where arid climate dominates, the IA caused by A. flavus is more common than that caused by A. fumigatus since A. flavus has the ability to survive higher temperatures.[5,7]The first line drug for the medical treatment and prophylaxis of IA is voriconazole.[8,9] Voriconazole belongs to the subclass of triazoles which is a lanosterol 14 alpha-demethylase inhibitor.[10]. On the basis of recent studies, voriconazole-resistant A.flavus demonstrates a wide range of MDR1, 2, 4overexpression as compared with the wild-type strain.[17] RNA interference (RNAi) is a posttranscriptional gene silencing (PTGS) phenomenon by which RNA molecules knock down essential genes responsible for vital as well as virulence factors.[18,19] The RNAi machinery is mediated by short 21–25 nucleotide small-interfering RNAs (siRNA) which are the products of a double-stranded RNA (dsRNA) by the action of RNase III-like enzyme called dicer.[20,21,22] These siRNAs will be incorporated into a multi-protein siRNA complex known as the RNA-induced silencing complex (RISC).[23,24] The RISC complex uses the incorporated siRNAs to target and degrade homologous messenger RNAs (mRNAs), leading to the silencing of the expression of corresponding endogenous genes.[25,26] A homology-dependent gene silencing phenomenon in fungi, termed “quelling”, was first demonstrated in ascomycete Neurospora crassa.[22] Recently, RNAi has been reported in several filamentous fungi like Aspergillus fumigatus and Aspergillus nidulans.[27,28] 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.[22] this study is an endeavor to delve into the silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant A.flavus as the target genes
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