Alteration in Abundance and Compartmentalization of Inflammation-Related miRNAs in Plasma After Intracerebral Hemorrhage
Background and Purpose—We tested the hypothesis that circulating microRNAs (miRNAs) present in plasma might display a specific signature in patients with intracerebral hemorrhage.
Methods—Global miRNA profiles were determined with the Agilent Human miRNA Microarray platform, and validated by quantitative polymerase chain reaction.
Results—A subset of 30 miRNAs were selectively upregulated in both male and female patients with intracerebral hemorrhage. Network analysis revealed that the most significantly overrepresented biological process associated with these miRNAs was inflammation. Unlike the plasma miRNAs in healthy controls, which were detected exclusively in the microvesicle fraction, the deregulated plasma miRNAs after intracerebral hemorrhage were present in both the microvesicle and the supernatant fractions.
Conclusions—Intracerebral hemorrhage altered both the abundance and the compartmentalization of several inflammation-related miRNAs in plasma.
MicroRNAs (miRNAs) are a class of short RNA molecules that have fundamental roles in post-transcriptional regulation of gene expression.1,2 The majority of miRNAs are present intracellularly; however, mounting evidence has suggested that miRNAs can also be detected in circulation (eg, in plasma or serum), and these cell-free miRNAs are relatively stable.2 Profiling studies have revealed that unique patterns of changed circulating miRNAs are associated with different diseases, including cancer, coronary heart disease, and diabetes mellitus.3–5 Previous studies on miRNAs and stroke mainly focused on ischemic stroke,6 whereas little is known about the relationships between circulating miRNAs and spontaneous intracerebral hemorrhage (ICH). In this study, we tested the hypothesis that circulating miRNAs from patients with ICH might display a characteristic change on both concentration and compartmentalization in the plasma.
Materials and Methods
This study was approved by the hospital Human Ethics Committee, and informed consents were obtained before recruitment. Patients with spontaneous ICH (n=15) or ischemic stroke (n=16) and 8 healthy volunteers were recruited (Table I in the online-only Data Supplement). A separate cohort of 12 controls and 11 patients with ICH were used for the validation experiments (Table II in the online-only Data Supplement). An expanded Methods is available in the online-only Data Supplement.
The complete microarray data are accessible at the Gene Expression Omnibus (accession number GSE43618). A total of 300 miRNA species were detected by the microarray. In male patients, we identified 70 miRNA species that were changed ≥2-fold as compared with healthy controls (48 upregulated and 22 downregulated). In female patients, 42 miRNAs (37 upregulated and 5 downregulated) were changed ≥2-fold (Figure 1A; Table III in the online-only Data Supplement). Cluster analysis of these altered miRNAs revealed that in both men and women, patients and controls displayed distinct miRNA profiles (Figure 1B). To minimize the potential bias introduced by sex differences, we excluded those miRNAs that were differentially altered in men and women from further analysis. As a result, we have identified 30 miRNAs that were upregulated in a sex-independent manner in patients with ICH (Figure 1B; Table IV in the online-only Data Supplement). To clarify whether the altered circulating miRNA profile in ICH was unique, we also analyzed the plasma miRNA from 16 ischemic stroke patients with a similar risk profile (Tables I and III in the online-only Data Supplement). We found that among the 30 upregulated miRNAs in ICH, 13 miRNAs (43%) were not changed in ischemic patients (Figure 1C; Table IV in the online-only Data Supplement).
To confirm the microarray data, we selected miR-29b, miR-365, miR-27a, miR-150*, miR-34c-3p, and miR-24, and performed quantitative polymerase chain reaction assays. Using standard curve method, we showed that the quantitative polymerase chain reaction results exhibited the same trend as the microarray data (Figure 2). We further validated these findings in a separate validation cohort (Table II in the online-only Data Supplement). We showed that the plasma levels of miR-27a, miR-365, miR-150*, and miR-34c-3p were significantly increased in patients with ICH, whereas the change of miR-29b did not reach a significance (Figure I in the online-only Data Supplement). Then we performed functional annotation analysis and found that the most significantly over-represented biological processes in association with the deregulated miRNAs were inflammatory disease and inflammatory response (Figure II and Table V in the online-only Data Supplement).
To examine whether disease conditions may disturb miRNA compartmentalization in the plasma, we isolated microvesicles and analyzed the relative abundance of miRNAs in the particulate and supernatant fractions with quantitative polymerase chain reaction. In plasma from healthy controls, miRNAs could not be detected in the supernatant fraction but were present exclusively in the microvesicle fraction (Figure 3A). In contrast, these miRNAs were present in both the supernatant and the microvesicle fractions in the plasma from stroke patients (Figure 3B).
In this study, we showed that the circulating miRNA levels in patients with ICH exhibited a unique pattern of change as compared with healthy controls. To eliminate possible effects of confounding factors, such as age and blood pressure, we also included an ischemic stroke group with comparable clinical parameters as ICH. We found that 13 deregulated miRNAs were unique to ICH and not found to be altered in ischemic stroke. These results suggested that the altered circulating miRNA profile after ICH was unlikely to be because of clinical complications caused by neurological deficits. Moreover, we confirmed the trend of changes in circulating miRNAs after ICH in a separate validation cohort with similar clinical conditions (except for the higher systolic blood pressure in ICH subjects). Likewise, a recent microarray study showed that multiple circulating miRNAs are differentially expressed in ICH patients with or without secondary hematoma enlargement.7 Bioinformatics analysis of the present data suggested that the upregulated miRNA species were specifically enriched in biological processes of inflammatory disease and inflammation response. This result is consistent with a previous study on mRNA expression of inflammation-related genes, showing that a complex inflammatory response is present after experimental ICH.8 A limitation of the present study, however, was that we could not completely exclude the potential impacts of certain confounding factors, such as systolic blood pressure on circulating miRNAs.
Recent studies have provided evidence that miRNAs in the plasma are encapsulated in membranous microvesicles and thereby protected from degradation.2 Using quantitative polymerase chain reaction, we confirmed that in healthy controls, the selected miRNAs were only detectable in the microvesicle fraction of plasma. Interestingly, in the ICH plasma, we found that the upregulated miRNAs existed in both the microvesicle and the supernatant fractions, suggesting that the disease condition had impacts on both the total concentration of miRNAs and their association with microvesicles in plasma. It should be noted that the physical form of circulating miRNAs in plasma is still under debate. For example, using different techniques, Arroyo et al9 provided evidence that the majority of circulating miRNAs cofractionated with protein complexes but not microvesicles, raising the possibility that cells might be able to secrete miRNA molecules via different routes.
The origin of the increased miRNAs in ICH plasma remains to be identified. Stroke patients are associated with a systemic inflammatory status.10,11 Interestingly, it is noted that a number of the upregulated miRNAs in ICH plasma are inflammatory cell-derived miRNAs, including miR-150, miR-365, miR-30c, miR-27a, miR-574-5p, miR-130a, and miR-423.12–14 Taken together, it is likely that the upregulated circulating mRNAs after ICH may be a result of increased microvesicle release from inflammatory cells.13,14 In addition, the circulating miRNAs can also be released from mechanically destructed blood vessel cells and injured neural cells caused by ICH.15
Sources of Funding
This work was partially supported by grants from National 973 Basic Research Program (2010CB732605, 2011CB503906) and Natural Science Foundation of China (#81271269, 81070224).
- Received January 17, 2013.
- Revision received February 21, 2013.
- Accepted March 1, 2013.
- © 2013 American Heart Association, Inc.
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