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Establishment of microRNA, transcript and protein regulatory networks in Alport syndrome induced pluripotent stem cells.

ABSTRACT: Alport syndrome (AS) is an inherited progressive disease caused by mutations in genes encoding for the ?3, ?4 and ?5 chains, which are an essential component of type IV collagen and are required for formation of the glomerular basement membrane. However, the underlying etiology of AS remains largely unknown, and the aim of the present study was to examine the genetic mechanisms in AS. Induced pluripotent stem cells (iPSCs) were generated from renal tubular cells. The Illumina HiSeq™ 2000 system and iTRAQ?coupled 2D liquid chromatography?tandem mass spectrometry were used to generate the sequences of microRNAs (miRNAs), transcripts and proteins from AS?iPSCs. Integration of miRNA, transcript and protein expression data was used to construct regulatory networks and identify specific miRNA targets amongst the transcripts and proteins. Relative quantitative proteomics using iTRAQ technology revealed 383 differentially abundant proteins, and high?throughput sequencing identified 155 differentially expressed miRNAs and 1,168 differentially expressed transcripts. Potential miRNA targets were predicted using miRanda, TargetScan and Pictar. All target proteins and transcripts were subjected to network analysis with miRNAs. Gene ontology analysis of the miRNAs and their targets revealed functional information on the iPSCs, including biological process and cell signaling. Kyoto Encyclopedia of Genes and Genomes pathways analysis revealed that the transcripts and proteins were primarily enriched in metabolic and cell adhesion molecule pathways. In addition, the network maps identified hsa?miRNA (miR)?4775 as a prominent miRNA that was associated with a number of targets. Similarly, the prominent ELV?like protein 1?A and epidermal growth factor receptor (EGFR)?associated transcripts were identified. Reverse transcription?quantitative polymerase chain reaction analysis was used to confirm the upregulation of hsa?miR?4775 and EGFR. The integrated approach used in the present study provided a comprehensive molecular characterization of AS. The results may also further understanding of the genetic pathogenesis of AS and facilitate the identification of candidate biomarkers for AS.

SUBMITTER: Chen W

PROVIDER: S-EPMC6297794 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Establishment of microRNA, transcript and protein regulatory networks in Alport syndrome induced pluripotent stem cells.

Chen Wenbiao W Tang Donge D Dai Yong Y Diao Hongyan H

Molecular medicine reports 20181120 1

Alport syndrome (AS) is an inherited progressive disease caused by mutations in genes encoding for the α3, α4 and α5 chains, which are an essential component of type IV collagen and are required for formation of the glomerular basement membrane. However, the underlying etiology of AS remains largely unknown, and the aim of the present study was to examine the genetic mechanisms in AS. Induced pluripotent stem cells (iPSCs) were generated from renal tubular cells. The Illumina HiSeq™ 2000 system ...[more]

PMID: 30483741

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Project description:Nakajo-Nishimura syndrome is a proteasome-associated autoinflammatory syndrome with a distinct homozygous mutation in the PSMB8 gene encoding an inducible β5i subunit of the immunoproteasome. Although it is considered that immunoproteasome dysfunction causes cellular stress and contributes to the production of inflammatory cytokines and chemokines, its detailed mechanism is still unknown. On the other hand, hereditary autoinflammatory diseases are considered as a good target for the analyses using induced pluripotent stem cells, whose differentiation systems to the innate immune cells such as neutrophils and monocytes have been established. Therefore, to elucidate the pathogenesis of Nakajo-Nishimura syndrome, we attempted in vitro disease modeling using patient-derived induced pluripotent stem cells. For analyses, isogenic control cells in which the responsible mutation was repaired and another pair of healthy embryonic stem cells and isogenic mutant cells in which the same mutation was introduced had also been prepared with genetic engineering. By comparing a pair of isogenic cells with the wild-type and the mutant PSMB8 gene after differentiation into monocytes and immortalization to synchronize their differentiation stages, the reduction of immunoproteasome enzyme activity and increased cytokine and chemokine production in the mutant cells without stimulation or with interferon-γ plus tumor necrosis factor-α stimulation were observed, and therefore, the autoinflammatory phenotype was successfully reproduced. Decreased cytokine production was observed by the addition of antioxidants as well as inhibitors for Janus kinase and p38-mitogen-activated protein kinase. At the same time, the increased production of reactive oxygen species and phosphorylation of both signal transducers and activator of transcription 1 and p38-mitogen-activated protein kinase were detected without stimulation. Notably, an antioxidant specifically decreased the constitutive phosphorylation of signal transducers and activator of transcription 1. These results indicate the usefulness of a disease modeling using pluripotent stem cell-derived cells in clarification of the pathomechanism and discovery of new therapeutic drugs for Nakajo-Nishimura syndrome and related proteasome-associated autoinflammatory syndromes.

Dataset Information

Establishment of microRNA, transcript and protein regulatory networks in Alport syndrome induced pluripotent stem cells.

Publications

Establishment of microRNA, transcript and protein regulatory networks in Alport syndrome induced pluripotent stem cells.

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