Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis - PubMed (original) (raw)

doi: 10.1371/journal.pone.0032999. Epub 2012 Mar 7.

Tobias Mollnow, Brenda Bongaerts, Natalia Elfimova, David Vargas Cardenas, Katharina Berger, Henning Zimmermann, Alexander Koch, Mihael Vucur, Mark Luedde, Claus Hellerbrand, Margarete Odenthal, Christian Trautwein, Frank Tacke, Tom Luedde

Affiliations

Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis

Christoph Roderburg et al. PLoS One. 2012.

Abstract

Background and aims: Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.

Methods: We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.

Results: The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.

Conclusion: Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Systematic profiling of serum miRNA levels in patients with distinct entities of liver cirrhosis.

(A) Microarray analysis for miRNA levels was performed using RNA extracts from serum of four healthy subjects as control and patients with liver cirrhosis of different etiologies and stages of disease (for detailed clinical parameters see Data S1). The fifteen miRNAs with strongest up- or down-regulation are depicted. Significantly regulated miRNAs are highlighted in bold type. (B) Hierarchical cluster analysis of the significantly regulated miRNAs miR-513-3p; miR-571; miR-652; bright green: under-expression; black: no change; bright red: over-expression.

Figure 2

Figure 2. Serum levels of miR-513-3p, miR-571 and miR-652 are significantly altered in the serum of patients with chronic liver disease and liver cirrhosis.

(A) Serum levels of the three significantly regulated miRNAs (miR-513-3p; miR-571; miR-652) were analyzed by qPCR in a collective of 17 healthy controls and 67 patients with chronic liver disease and liver cirrhosis. Results are depicted as box plot. The thick line represents the median of relative expression. (B) Receiver operating characteristic (ROC) curve analysis displaying the diagnostic power in predicting cirrhosis of these miRNAs when analyzed as single markers (area under the curve (AUC) 0.87; 0.91; 0.75). (C) ROC curve analysis displaying the diagnostic power in predicting cirrhosis of different combinations of these miRNAs (AUC 0.91; 0.962; 0.9676). **P<0.001.

Figure 3

Figure 3. Correlation of miRNA serum levels with Child-Pugh class and etiology of liver disease.

(A) Microarray data of the serum of eight patients with alcoholic liver cirrhosis at different stages of disease (Child-Pugh A and Child-Pugh C) in a hierarchical cluster analysis; bright green: under-expression; black: no change; bright red: over-expression. (B) Hierarchical cluster analysis of the significantly regulated miRNAs; bright green: under-expression; black: no change; bright red: over-expression. (C) Serum levels of miR-513-3p, miR-571 and miR-652 in the patients with alcoholic liver cirrhosis were analyzed by qPCR. Results are depicted as box plot. The thick line represents median of relative expression. (D) Microarray data of serum of the serum of four patients with ethanol-toxic liver cirrhosis (Child-Pugh A) and the four hepatitis C related cirrhosis (Child-Pugh A) in a hierarchical-cluster-analysis; bright green: under-expression; black: no change; bright red: over-expression. (E) Hierarchical cluster analysis of the significantly regulated miRNAs; bright green: under-expression; black: no change; bright red: over-expression. (F) Serum levels of miR-513-3p, miR-571 and miR-652 in the patients with alcoholic liver cirrhosis (Child-Pugh A) and hepatitis C related cirrhosis (Child-Pugh A) were analyzed by qPCR. Results are depicted as box plot. The thick line represents median of relative expression. **P<0.01.

Figure 4

Figure 4. Serum levels of miR-571 and miR-652 reflect their specific regulation in distinct organs and cell compartments involved in the pathogenesis of liver cirrhosis.

(A) Expression of miR-513-3p, miR-571 and miR-652 was analyzed by qPCR in liver samples of patients with liver cirrhosis (n = 13) and livers from patients without chronic liver disease (n = 10) as control. (B) Expression of miR-513-3p, miR-571 and miR-652 was analyzed by qPCR in MACS-sorted monocytes of patients with liver cirrhosis (n = 19) and healthy donors (n = 19) as control. (C) Expression of miR-513-3p, miR-571 and miR-652 was analyzed by qPCR in lymphocytes of patients with liver cirrhosis (n = 11) and healthy donors (n = 6) as control. Results are depicted as dot plot with each dot representing one individual patient. The line represents the mean of relative expression. (D) qPCR analysis of miR-571 and miR-652 expression in human primary hepatocytes, HSCs and monocytes, respectively. Results are depicted as mean, error bars denote SEM. *P<0.05, ***P<0.001.

Figure 5

Figure 5. miR-571 and miR-652 integrate pro-fibrotic and inflammatory signals in human HSCs and monocytes.

(A) Expression of miR-571 and miR-652 in primary human HSCs in response to stimulation with TGF-β for 48 h was measured by qPCR. Results are depicted as dot plot with each dot representing one individual patient. The line represents the mean of relative expression. (B) We stimulated U937 cells with PMA into a monocytic differentiation. miR-571 and miR-652 expression levels in response to stimulation with LPS for 48 h were measured by qPCR. Results are depicted as dot plot with each dot representing one individual patient. The line represents the mean of relative expression. (C) Expression of CREBBP was analyzed by qPCR in liver samples of patients with liver cirrhosis (n = 13) and non-liver diseased donors (n = 10) as control. Results are depicted as mean, error bars denote SEM. (D) HuH-7 cells and LX-2 cells were transfected with indicated amounts of miR-571 mimic. Expression of CREBBP was determined after two days of transfection by qPCR. Results are depicted as mean, error bars denote SEM. (E) Activation of stellate cells was determined by qPCR-based analysis of SMA- and Col1A1-expression in LX-2 cells. **P<0.01, ***P<0.001.

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