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Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

ABSTRACT: MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-?, Hedgehog signaling, adherens junctions, TGF-?, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.

SUBMITTER: Poe AJ

PROVIDER: S-EPMC7650592 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

Poe Adam J AJ Kulkarni Mangesh M Leszczynska Aleksandra A Tang Jie J Shah Ruchi R Jami-Alahmadi Yasaman Y Wang Jason J Kramerov Andrei A AA Wohlschlegel James J Punj Vasu V Ljubimov Alexander V AV Saghizadeh Mehrnoosh M

Cells 20200926 10

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to c ...[more]

PMID: 32993109

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Project description:Purpose: Most studies conducted towards understanding the differences among the adult sources of MSCs (BM-MSC and AD-MSC) and UC-MSCs have focused on their phenotypic properties which cannot provide an idea of the functional characteristics changing at the molecular level. Even NGS studies done before, each each study focuses on a different profiling methodology, which makes it difficult to establish a molecular equivalency among the various datasets. We undertake an integrative omics comparison between UC-MSC and two other popular sources of adult MSC (BM-MSC and AD-MSC), with the reasoning that a joint modeling of the changes occurring at the various functional levels will provide greater in-depth insight into the molecular heterogeneity related to different sources of MSCs. Methods: human Mesenchymal Stem Cell (MSC) mRNA cultured under basal conditions in Xeno-free media were subjected to deep sequencing. Three different MSC sourcers : adipose (AD-MSC), bone-marrow (BM-MSC) and Wharton-jelly umbilical cord (UC-MSC) were used and three donors from each tissues source along with three biological replicates for each donor were sequenced using the Illumina PE 150 platform. The sequence reads that passed quality filters Q30≥ 80% were analyzed using DESeq2. Results: Using an optimized data analysis workflow, we identified 24,804 transcripts among the three different sources of MSCs. Among these 2127 transcripts (~10%) were found to be differentially expressed among the three sources of MSC, with a\Log2FC| ≥2 and FDR <0.05. Hierarchical clustering of differentially expressed genes uncovered significant differences among the neonatal source (UC-MSC) and adult-MSCs (AD-MSC and BM-MSC). Altered gene expression profiles among the sources was also confirmed using a proteomics study of the same three sources of MSC with the same donors. Further, secretory immune-regulatory molecules which were confirmed to be differentially expressed among the MSC sources using the integrated transcriptomics-proteomics approach was validated using a Luminex cytokine profiling study. Conclusions: Our study represents the first detailed integrated analysis of differential gene expression profiling among source-specific human Mesenchymal Stem Cells using a multi-omics approach to validate the observed transcriptomic changes at both the translational as well as secretory levels. Our results offer a comhrehensive evaluation of the differences in immune signaling that exists among the different sourcrs of MSC based on their tissue of origina nd which subsequently plays a decisive role in determining the "regenrative siganture" of these MSCs based on the match between "immune-signature" and "disease type".

Dataset Information

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

Publications

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

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