Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients

Lange, Tim; Stracke, Sylvia; Rettig, Rainer; Lendeckel, Uwe; Kuhn, Jana; Schlüter, Rabea; Rippe, Volkhard; Endlich, Karlhans; Endlich, Nicole

doi:10.1371/journal.pone.0183435

Cited by 97 publications

(68 citation statements)

References 51 publications

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“…As these results suggested transcriptional regulation of miRNA expression in L. pneumophila infection, as opposed to changes in miRNA processing, the pri-miRNA expression levels of miR-146a, miR-155, miR-125b, and miR-221 were analyzed by qPCR. pri-mir-16 served as an endogenous control (33,34). Upregulation of pri-mir-155 and -146a ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

Herkt

Caffrey

Surmann

et al. 2020

mBio

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Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages. IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.

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Section: Resultsmentioning

confidence: 99%

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

Herkt

Caffrey

Surmann

et al. 2020

mBio

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“…According to the literature, 12 miRNAs and one small RNA (U6 snRNA) were selected for stability analysis (Table 1) [8,[48][49][50][51][52][53][54][55]. 2.6.…”

Section: Candidate Rg Selectionmentioning

confidence: 99%

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

et al. 2020

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Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and ΔCt method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields.

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“…Yu et al found that the renal tubule-derived urinary exosomal miR-200b could regulate the progression of renal fibrosis, eventually leading to CKD [126]. Lange et al concluded that miR-16 derived from urinary exosomes was the most stable endogenous reference gene in patients with CKD [127].…”

Section: The Role Of Exosomes In Vascular Aging Related Kidney Diseasesmentioning

confidence: 99%

Roles and Functions of Exosomal Non-coding RNAs in Vascular Aging

Ni¹,

Lin²,

Zhan³

et al. 2020

Aging and disease

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Aging is a progressive loss of physiological integrity and functionality process which increases susceptibility and mortality to diseases. Vascular aging is a specific type of organic aging. The structure and function changes of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are the main cause of vascular aging, which could influence the threshold, process, and severity of vascular related diseases. Accumulating evidences demonstrate that exosomes serve as novel intercellular information communicator between cell to cell by delivering variety biologically active cargos, especially exosomal non-coding RNAs (ncRNAs), which are associated with most of aging-related biological and functional disorders. In this review, we will summerize the emerging roles and mechanisms of exosomal ncRNAs in vascular aging and vascular aging related diseases, focusing on the role of exosomal miRNAs and lncRNAs in regulating the functions of ECs and VSMCs. Moreover, the relationship between the ECs and VSMCs linked by exosomes, the potential diagnostic and therapeutic application of exosomes in vascular aging and the clinical evaluation and treatment of vascular aging and vascular aging related diseases will also be discussed.

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Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients

Cited by 97 publications

References 51 publications

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

Roles and Functions of Exosomal Non-coding RNAs in Vascular Aging

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