Quantitative Analysis of miRNA Expression in Epithelial Cells and Tissues

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

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Clustered MicroRNAs of the Epstein-Barr Virus Cooperatively Downregulate an Epithelial Cell-Specific Metastasis Suppressor

Journal of Virology ◽

10.1128/jvi.03189-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2684-2697 ◽

Cited By ~ 51

Author(s):

Teru Kanda ◽

Mamiko Miyata ◽

Makoto Kano ◽

Satoru Kondo ◽

Tomokazu Yoshizaki ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mirna Expression ◽

Epstein Barr Virus ◽

Epithelial Differentiation ◽

Metastasis Suppressor ◽

Barr Virus ◽

Epstein Barr ◽

Differentiation Marker ◽

Bart Mirnas

ABSTRACTThe Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Althoughin vitroreporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering thatNDRG1encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesisin vivo.IMPORTANCEEBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

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Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

Molecular and Cellular Biology ◽

10.1128/mcb.00553-18 ◽

2019 ◽

Vol 39 (14) ◽

Cited By ~ 7

Author(s):

Stephanie Muenchau ◽

Rosalie Deutsch ◽

Ines J. de Castro ◽

Thomas Hielscher ◽

Nora Heber ◽

...

Keyword(s):

Epithelial Cells ◽

Mirna Expression ◽

Barrier Function ◽

Intestinal Epithelial Cells ◽

Hypoxia Inducible Factor ◽

Intestinal Epithelial ◽

Low Oxygen ◽

Direct Role ◽

Hypoxic Conditions ◽

Barrier Formation

ABSTRACT Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

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Faecal microRNAs: indicators of imbalance at the host-microbe interface?

Beneficial Microbes ◽

10.3920/bm2017.0013 ◽

2018 ◽

Vol 9 (2) ◽

pp. 175-183 ◽

Cited By ~ 18

Author(s):

G.M. Moloney ◽

M.F. Viola ◽

A.E. Hoban ◽

T.G. Dinan ◽

J.F. Cryan

Keyword(s):

Epithelial Cells ◽

Gut Microbiota ◽

Antibiotic Treatment ◽

Mirna Expression ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Sprague Dawley ◽

Faecal Pellets ◽

Non Invasive ◽

Bodily Fluids

The enteric microbiota is characterised by a balance and composition that is unique to the host. It is important to understand the mechanisms through which the host can maintain the composition of the gut microbiota. MicroRNAs (miRNA) are implicated in intercellular communication and have been isolated from bodily fluids including stool. Recent findings suggest that miRNA produced by the host’s intestinal epithelial cells (IECs) participate in shaping the microbiota. To investigate whether miRNA expression was influenced by the gut microbiota we measured the expression of miRNAs expressed by intestinal epithelial cells in faeces. Specifically, we measured miRNA expression in faeces from germ-free (GF) and conventional mice and similarly in a rat model of antibiotic-mediated depletion of the gut microbiota control rats. In adult male GF and conventional mice and adult Sprague Dawley (SD) rats were treated with a combination of antibiotics for 8 weeks; total RNA was extracted from faecal pellets taken at week 0, 2, 4, 6 week 8 and the expression of let-7b-3p, miR-141-3p, miR-200a-3p and miR-1224-5p (miRNAs known to be expressed in IECs) were measured relative to U6 at each time point using qRT-PCR. In GF animals the expression of let-7b, miR-141 and miR-200a in faeces was lower compared to conventional mice. Following antibiotic-mediated depletion of gut microbiota, rats showed two divergent profiles of miRNA expression. Following two weeks of antibiotic treatment, the expression of let-7b and miR-1224 dropped significantly and remained low for the remainder of the study. The expression of miR-200a and miR-141 was significantly higher at week 2 than before antibiotic treatment commenced. Subsequently, the expression of miR-200a and miR-141 decreased at week 4 and continued to decrease at week 6. This data demonstrates that miRNAs can be used as an independent, non-invasive marker of microbial fluctuations along with gut pathology in the intestine.

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Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00401.2011 ◽

2012 ◽

Vol 302 (11) ◽

pp. G1282-G1291 ◽

Cited By ~ 8

Author(s):

Moiz A. Charania ◽

Saravanan Ayyadurai ◽

Sarah A. Ingersoll ◽

Bo Xiao ◽

Emilie Viennois ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mirna Expression ◽

Target Genes ◽

Immune Cell ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Cell Types ◽

Intestinal Epithelial ◽

Small Noncoding Rnas

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.

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Chronic Administration of Δ9-Tetrahydrocannabinol Induces Intestinal Anti-Inflammatory MicroRNA Expression during Acute Simian Immunodeficiency Virus Infection of Rhesus Macaques

Journal of Virology ◽

10.1128/jvi.01754-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1168-1181 ◽

Cited By ~ 37

Author(s):

Lawrance C. Chandra ◽

Vinay Kumar ◽

Workineh Torben ◽

Curtis Vande Stouwe ◽

Peter Winsauer ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Epithelial Cells ◽

Mirna Expression ◽

Intestinal Inflammation ◽

Rhesus Macaques ◽

Chronic Administration ◽

Oxygen Species ◽

Immunodeficiency Virus ◽

Gastrointestinal Inflammation ◽

Anti Inflammatory

ABSTRACTRecreational and medical use of cannabis among human immunodeficiency virus (HIV)-infected individuals has increased in recent years. In simian immunodeficiency virus (SIV)-infected macaques, chronic administration of Δ9-tetrahydrocannabinol (Δ9-THC) inhibited viral replication and intestinal inflammation and slowed disease progression. Persistent gastrointestinal disease/inflammation has been proposed to facilitate microbial translocation and systemic immune activation and promote disease progression. Cannabinoids including Δ9-THC attenuated intestinal inflammation in mouse colitis models and SIV-infected rhesus macaques. To determine if the anti-inflammatory effects of Δ9-THC involved differential microRNA (miRNA) modulation, we profiled miRNA expression at 14, 30, and 60 days postinfection (days p.i.) in the intestine of uninfected macaques receiving Δ9-THC (n= 3) and SIV-infected macaques administered either vehicle (VEH/SIV;n= 4) or THC (THC/SIV;n= 4). Chronic Δ9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60 days p.i. At 60 days p.i., ∼28% of miRNAs showed decreased expression in the VEH/SIV group compared to none showing decrease in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149, and miR-187, previously been shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator, was confirmed as a direct miR-99b target. A significant increase in NOX4+crypt epithelial cells was detected in VEH/SIV macaques compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. These results support a role for differential miRNA induction in THC-mediated suppression of intestinal inflammation. Whether similar miRNA modulation occurs in other tissues requires further investigation.IMPORTANCEGastrointestinal (GI) tract disease/inflammation is a hallmark of HIV/SIV infection. Previously, we showed that chronic treatment of SIV-infected macaques with Δ9-tetrahydrocannabinol (Δ9-THC) increased survival and decreased viral replication and infection-induced gastrointestinal inflammation. Here, we show that chronic THC administration to SIV-infected macaques induced an anti-inflammatory microRNA expression profile in the intestine at 60 days p.i. These included several miRNAs bioinformatically predicted to directly target CXCL12, a chemokine known to regulate lymphocyte and macrophage trafficking into the intestine. Specifically, miR-99b was significantly upregulated in THC-treated SIV-infected macaques and confirmed to directly target NADPH oxidase 4 (NOX4), a reactive oxygen species generator known to damage intestinal epithelial cells. Elevated miR-99b expression was associated with a significantly decreased number of NOX4+epithelial cells in the intestines of THC-treated SIV-infected macaques. Overall, our results show that selective upregulation of anti-inflammatory miRNA expression contributes to THC-mediated suppression of gastrointestinal inflammation and maintenance of intestinal homeostasis.

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Hypoxic environment promotes barrier formation in human intestinal epithelial cells through regulation of miRNA-320a expression

10.1101/483206 ◽

2018 ◽

Author(s):

Stephanie Muenchau ◽

Rosalie Deutsch ◽

Thomas Hielscher ◽

Nora Heber ◽

Beate Niesler ◽

...

Keyword(s):

Epithelial Cells ◽

Mirna Expression ◽

Barrier Function ◽

Intestinal Epithelial Cells ◽

Hypoxia Inducible Factor ◽

Intestinal Epithelial ◽

Low Oxygen ◽

Direct Role ◽

Hypoxic Conditions ◽

Barrier Formation

AbstractIntestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions are on one hand fundamental for the survival of the commensal microbiota, and on the other hand, favor the formation of a selective semipermeable barrier allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified miRNA-320a as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing we could demonstrate that expression of this miRNA was HIF-dependent. Importantly, using over-expression and knock-down approaches of miRNA-320a we could confirm its direct role in the regulation of barrier functions in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

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