Suppression of Foxo3-Gatm by miR-132-3p Accelerates Cyst Formation by Up-Regulating ROS in Autosomal Dominant Polycystic Kidney Disease.

Accumulation of reactive oxygen species (ROS) is associated with the development of various diseases. However, the molecular mechanisms underlying oxidative stress that lead to such diseases like autosomal dominant polycystic kidney disease (ADPKD) remain unclear. Here, we observed that oxidative stress markers were increased in Pkd1f/f:HoxB7-Cre mice. Forkhead transcription factors of the O class (FOXOs) are known key regulators of the oxidative stress response, which have been observed with the expression of FoxO3a in an ADPKD mouse model in the present study. An integrated analysis of two datasets for differentially expressed miRNA, such as miRNA sequencing analysis of Pkd1 conditional knockout mice and microarray analysis of samples from ADPKD patients, showed that miR-132-3p was a key regulator of FOXO3a in ADPKD. miR-132-3p was significantly upregulated in ADPKD which directly targeted FOXO3 in both mouse and human cell lines. Interestingly, the mitochondrial gene Gatm was downregulated in ADPKD which led to a decreased inhibition of Foxo3. Overexpression of miR-132-3p coupled with knockdown of Foxo3 and Gatm increased ROS and accelerated cyst formation in 3D culture. This study reveals a novel mechanism involving miR-132-3p, Foxo3, and Gatm that is associated with the oxidative stress that occurs during cystogenesis in ADPKD.

Molecular typing of Cyclospora cayetanensis in produce and clinical samples using targeted enrichment of complete mitochondrial genomes and next-generation sequencing.

BACKGROUND: Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS: We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS: This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS: Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models.

Autosomal polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by the development of numerous fluid-filled cysts in both kidneys. We investigated miRNA-mediated regulatory systems and networks that play an important role during cystogenesis through integrative analysis of miRNA- and RNA-seq using two ADPKD mouse models (conditional Pkd1- or Pkd2-deficient mice), at three different time points (P1, P3, and P7). At each time point, we identified 13 differentially expressed miRNAs (DEmiRs) and their potential targets in agreement with cyst progression in both mouse models. These targets were involved in well-known signaling pathways linked to cystogenesis. More specifically, we found that the actin cytoskeleton pathway was highly enriched and connected with other well-known pathways of ADPKD. We verified that miR-182-5p regulates actin cytoskeleton rearrangement and promotes ADPKD cystogenesis by repressing its target genes-Wasf2, Dock1, and Itga4-in vitro and in vivo. Our data suggest that actin cytoskeleton may play an important role in renal cystogenesis, and miR-182-5p is a novel regulator of actin cytoskeleton and cyst progression. Furthermore, this study provides a systemic network of both key miRNAs and their targets associated with cyst growth in ADPKD.