Background: MicroRNAs (miRNAs) are endogenous small RNAs of 21–25 nucleotides that can pair with sites in 3′ untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. We assessed the hypothesis that miRNAs may leak into the circulating blood from injured cells and thereby serve as biomarkers for identifying the injured cell type.
Methods: We used isoproterenol-induced myocardial injury in rats as a model and miRNA array analyses to identify candidate miRNAs specifically produced in the ventricles of the heart. Individual miRNA concentrations were measured by real-time reverse-transcription PCR. Plasma cardiac troponin I (cTnI) concentrations were measured with an ELISA.
Results: Array analyses revealed miR-208 to be produced exclusively in the heart, and we selected this miRNA as a possible biomarker of myocardial injury. Plasma concentrations of miR-208 increased significantly (P < 0.0001) after isoproterenol-induced myocardial injury and showed a similar time course to the concentration of cTnI, a classic biomarker of myocardial injury.
Conclusions: The plasma concentration of miR-208 may be a useful indicator of myocardial injury. Our results suggest that profiling of circulating miRNAs may help identify promising biomarkers of various pathologic conditions.
MicroRNAs (miRNAs)1 are endogenous small RNAs of 21–25 nucleotides that can pair at sites in the 3′ untranslated region in mRNAs of protein-coding genes to downregulate their expression (1)(2). miRNAs play important roles in various physiological and pathologic processes (3)(4)(5)(6)(7). More than 500 human miRNAs have been identified (8), and most human protein-coding genes are thought to be targeted by these miRNAs (9)(10)(11). miRNAs appear to function as a rheostat to fine-tune adjustments to protein output (12)(13).
The existence of miRNAs in various body fluids has recently been reported (14)(15)(16)(17). miR-141, which is abundantly present in the placenta, has been reported to be present in the maternal circulation and in varying amounts at different gestational ages (17). Circulating miRNAs have been shown to be useful as diagnostic biomarkers of various cancers (18)(19). Because the miRNA profiles appear to be cell specific (20)(21)(22)(23)(24), identification of the profile of increased miRNAs in circulating blood may help determine the type of injured cell.
In this study, we assessed the hypothesis that miRNAs may leak into circulating blood from injured cells and that these circulating miRNAs may be useful for identifying the injured cell type. We used an isoproterenol-induced myocardial-injury model (25)(26)(27)(28)(29) to confirm whether cardiac-specific miRNAs leak from injured cardiomyocytes into the circulating blood and whether they can be used to diagnose myocardial injury.
Materials and Methods
Male Sprague Dawley rats (250–350 g) were purchased from Japan SLC. The rats were housed in a temperature-controlled room on a 12-h light/12-h dark cycle and fed standard rat chow (with 5 g/kg NaCl; Clea Japan) and tap water ad libitum. The present study was conducted in accordance with the guidelines of the National Cardiovascular Center for the care and use of experimental animals.
Isoproterenol (Wako Pure Chemical Industries) was dissolved in saline and administered subcutaneously (320 mg/kg) (25)(26)(27)(28)(29). Blood was collected from the tail vein at 0, 3, 6, 12, and 24 h after the administration of isoproterenol. Samples were collected into tubes containing EDTA (Terumo Medical Corporation), and the plasma was isolated by centrifugation. Plasma samples collected for miRNA assay were mixed with an equal volume of the denaturing solution in the mirVana PARIS Kit (Ambion) and maintained at −80 °C until purification.
Plasma cardiac troponin I (cTnI) concentrations were measured with an ELISA kit (Life Diagnostics) according to the manufacturer’s protocol.
Animal models of renal infarction were produced in Sprague Dawley rats by ligating peripheral branches of the left renal artery. Plasma samples were collected before and at 6 h after the operative procedure. The occurrence of renal infarction was verified by inspection and by detection of an increase in the plasma concentration of aspartate aminotransferase.
Total RNA was extracted from tissues with TRIzol Reagent (Invitrogen) as described previously (30). Total plasma RNA was isolated with the mirVana PARIS Kit according to the manufacturer’s protocol. In brief, total RNA was purified from 200 μL of plasma and ultimately eluted into 100 μL of RNase-free water. RNA was then precipitated with ethanol in the presence of a polyacrylamide polymer solution (Ethachinmate; Nippon Gene) and resuspended in 20 μL of RNase-free water. The recovery of miRNA by this purification method was assessed with 32P-labeled synthetic miR-208; the mean (SD) purity was estimated at 85.5% (15.5%) (n = 9).
identification of cardiac-specific mirnas by taqman microrna array analysis
To identify cardiac-specific miRNAs, we profiled the production of 359 miRNAs in rat tissues of liver, muscle, kidney, and heart with the ABI TaqMan MicroRNA Array kit (Applied Biosystems) according to the manufacturer’s protocol. In brief, 1 μg of rat tissue–derived total RNA was reverse-transcribed with Megaplex RT primers (Megaplex RT Rodent Pool A), followed by a real-time PCR with TaqMan Rodent MicroRNA Array (panel A) performed on an Applied Biosystems 7900HT System. SDS software v2.3 and RQ Manager 1.2 (Applied Biosystems) were used to obtain the comparative threshold cycle (CT) value. U6 small nuclear RNA included in the TaqMan Rodent MicroRNA Array was used as an endogenous control. The percentages of miRNAs that were undetectable by this method for muscle, liver, heart, and kidney tissue were 37.6%, 43.3%, 34.0%, and 31.9%, respectively.
quantification of mirnas by real-time reverse-transcription pcr (rt-pcr)
Concentrations of individual miRNAs were measured with a TaqMan MicroRNA real-time RT-PCR kit (31) (Applied Biosystems) according to the manufacturer’s protocol. The reverse-transcription reaction was initiated with 1 μg of tissue-derived total RNA or with 10 μL of plasma-derived total RNA (from 100 μL of plasma). Negative controls were included with every real-time RT-PCR assay, and no amplification of the signal was observed when water was added instead of RNA or cDNA sample. The ABI Prism 7700 Sequence Detection System (Applied Biosystems) was used for amplification and detection. The CT value was obtained from the amplification plot with the aid of SDS software.
To evaluate RNA recovery from plasma samples and amplification, we simultaneously measured the concentration of 5S rRNA (internal calibrator) by real-time RT-PCR. Ten microliters of plasma-derived total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 3.75 μmol/L of random hexamer primers. We then amplified the cDNA by the real-time PCR in the presence of forward primer (5′-CGTCTGATCTCGGAAGCTAAGC-3′), reverse primer (5′-GGCGGTCTCCCATCCAA-3′), and FAM/MGB probe (5′-FAM-TCGGGCCTGGTTAGT-MGB-3′). The amplification plot and the CT value were obtained with the ABI Prism 7700 System and the SDS software, as described earlier.
To measure the absolute amount of miR-208, we conducted the real-time RT-PCR assay with known amounts of synthetic rat miR-208 (Integrated DNA Technologies). In the presence of 0.5 amol (CT = 35) to 50 fmol (CT = 17.5) of synthetic miR-208, we observed a linear correlation (r2 = 0.99) between the logarithm of the amount of input RNA and the CT value. miR-208 was reliably measured at a detection limit equivalent to a CT of 35. Of note is that miR-208 was not detected in the plasma of healthy rats at all; no signal was detected even after 45 cycles of real-time PCR. The amount of miRNA not detected after 45 cycles of a real-time PCR was regarded in the present study as a CT equivalent to 45. Relative concentrations of miRNAs are expressed as 235−CT. We set 35 as the baseline because the limit for reliably detecting synthetic miR-208 was 0.5 amol (CT = 35).
Unless otherwise indicated, the mean and SD were calculated. Results were evaluated by ANOVA, followed by post hoc testing with Bonferroni correction. Values obtained at baseline (0 h) were used as the reference and compared with those obtained at 3, 6, 12, and 24 h after isoproterenol administration. To assess whether the time courses of miR-208 and cTnI differed, we normalized these values and compared them by multivariate ANOVA. Statistical analyses were performed with the JMP statistical package (version 7.0; SAS Institute).
identification of cardiac-specific mirnas
Our goal was to determine whether myocardium-specific miRNA could serve as a potential biomarker of myocardial injury. For this purpose, myocardium-specific miRNAs needed to be identified. The miRNA array analyses of 359 species of miRNAs in the heart, kidney, liver, and muscle suggested that miR-208 and miR-490 are produced exclusively in the heart (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol55/issue11). The production of these 2 miRNAs was thoroughly investigated further (Fig. 1⇓ ). miR-490 production was detected not only in the heart but also in the small and large intestines, lung, adrenal gland, and stomach. On the other hand, miR-208 production was detected exclusively in the heart. We therefore chose miR-208 as a candidate biomarker of myocardial injury.
concentrations of plasma mirnas in a myocardial-injury model
We injected rats with isoproterenol and measured the plasma concentrations of cTnI, miR-208, miR-183, and 5S RNA (Fig. 2⇓ ). Because of its specific presence in the eye (retina) and absence in the heart (Fig. 1⇑ ), miR-183 was selected as a negative-control miRNA not derived from the heart. 5S rRNA was used as an internal calibrator because it appears to be present in all cells; 5S rRNA concentrations in the circulating blood were high and relatively constant.
As is shown in Fig. 2⇑ , cTnI concentrations were significantly increased 3 h after isoproterenol administration (P < 0.05, Student t-test with Bonferroni correction), indicating myocardial injury. Circulating concentrations of cTnI were still increased 24 h after administration. Plasma concentrations of miR-208, which were undetectable at baseline (CT > 45), were also significantly increased 3 h after administration (CT = 30–33; P < 0.005; Fig. 2⇑ ) and remained significantly increased until 12 h after administration. No significant difference in the time course was found between miR-208 and cTnI (P = 0.09, multivariate ANOVA). As expected, miR-183 remained almost undetectable in the plasma (CT = 31–36). The concentration of 5S RNA remained unchanged throughout the experiment.
To eliminate the possibility that the observed increase in plasma miR-208 was caused by nonspecific insult, we assessed plasma miR-208 concentrations in a renal-infarction model (Fig. 3⇓ ). At 6 h after the procedure, plasma aspartate aminotransferase activities were significantly higher in rats with renal artery ligation than in sham-operated rats, indicating the occurrence of renal infarction in this model (Fig. 3D⇓ ). In contrast, plasma concentrations of miR-208 remained undetectable (Fig. 3A⇓ ). Interestingly, plasma concentrations of miR-10a—an miRNA produced at relatively highly concentrations in the kidney—were significantly increased (Fig. 3B⇓ ). These observations suggest that nonspecific insult does not appreciably increase the circulating concentration of miR-208 and that renal infarction might be more precisely diagnosed on the basis of the plasma concentration of kidney-specific miRNAs.
plasma concentrations of mirnas in a cardiac-hypertrophy model
Next, we examined plasma miR-208 concentrations in rats with hypertensive cardiac hypertrophy. Salt-sensitive Dahl rats maintained on a high-salt diet (80 g/kg NaCl; Oriental Yeast) for 8 weeks showed high blood pressure with marked cardiac and renal hypertrophy, as well as high plasma concentrations of B-type natriuretic peptide (data not shown); however, the plasma concentration of miR-208 did not increase in these rats (data not shown), thus suggesting that the hypertrophy was not sufficient for miR-208 to leak out of cardiac myocytes.
Accumulating evidence suggests the usefulness of circulating miRNAs as stable blood-based biomarkers for detection of cancers (14)(18)(19). For example, measurement of serum concentrations of miR-141 for diagnosing prostate cancer has been reported (19). Moreover, miR-141 has recently been identified as a pregnancy-associated biomarker (17). Our data show that the plasma concentration of miR-208, which is produced exclusively in the heart, increases in isoproterenol-induced myocardial injury. The plasma concentration of miR-208 shows a good correlation with the plasma concentration of cTnI, a classic marker of myocardial injury (32). Thus, our results clearly support the hypothesis that miRNAs may leak out of injured cells into the circulating blood and thereby serve as markers for identifying the type of injured cell.
The relatively high abundance of many circulating miRNAs suggests potential biological roles for miRNAs as extracellular messengers mediating cell–cell communication (15)(33), although such a hypothesis requires further investigation. miRNAs in plasma have been reported to be remarkably stable. It is likely that some proportion of circulating miRNAs are secreted from the cells as exosomes (15) and that some miRNAs may leak out of disrupted cells and associate with other molecules, thereby becoming protected from degradation [e.g., in an RNA-induced silencing complex (RISC) (2)]. It is likely, as in the case of cTnI, that a RISC containing miRNA may leak from the cytosol of injured myocytes into the circulating blood.
The validity of circulating cTnI as a marker of isoproterenol-induced myocardial injury has been confirmed (34). In our present analysis, the plasma concentrations of cTnI and miR-208 were highly correlated and exhibited similar time courses (Fig. 2⇑ ). The next question is whether assessment of plasma miR-208 has clinical significance and whether it offers advantages over measurement of cTnI and/or cTnT. Cardiac troponin concentrations are known to be occasionally increased in end-stage renal disease, even in the absence of an acute coronary syndrome (35), because cardiac troponins are excreted from the kidney. Our preliminary assessment, however, indicated that plasma miRNA concentrations were not increased in bilaterally nephrectomized rats (see Fig. 1 in the online Data Supplement). Thus, plasma miR-208 may be superior to cTnI and/or cTnT for detecting myocardial injury in individuals with renal dysfunction. Moreover, it is likely that the limit of detection for plasma miR-208 is lower than for cTnI (as well as cTnT) because detection of miR-208 in plasma is based on PCR analysis. These possible advantages should be assessed in future studies in clinical settings involving humans.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Program for the Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation, Japan.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
↵1 Nonstandard abbreviations: miRNA, microRNA; cTnI, cardiac troponin I; CT, threshold cycle; RT-PCR, reverse-transcription PCR; RISC, RNA-induced silencing complex.
- © 2009 The American Association for Clinical Chemistry