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Keywords:

  • biomarker;
  • child;
  • Duchenne muscular dystrophy;
  • microRNA;
  • serum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information
Thumbnail image of graphical abstract

Creatine kinase has been utilized as a diagnostic marker for Duchenne muscular dystrophy (DMD), but it correlates less well with the DMD pathological progression. In this study, we hypothesized that muscle-specific microRNAs (miR-1, -133, and -206) in serum may be useful for monitoring the DMD pathological progression, and explored the possibility of these miRNAs as potential non-invasive biomarkers for the disease. By using real-time quantitative reverse transcription–polymerase chain reaction in a randomized and controlled trial, we detected that miR-1, -133, and -206 were significantly over-expressed in the serum of 39 children with DMD (up to 3.20 ± 1.20, 2−ΔΔCt): almost 2- to 4-fold enriched in comparison to samples from the healthy controls (less than 1.15 ± 0.34, 2−ΔΔCt). To determine whether these miRNAs were related to the clinical features of children with DMD, we analyzed the associations compared to creatine kinase. There were very good inverse correlations between the levels of these miRNAs, especially miR-206, and functional performances: high levels corresponded to low muscle strength, muscle function, and quality of life. Moreover, by receiver operating characteristic curves analyses, we revealed that these miRNAs, especially miR-206, were able to discriminate DMD from controls. Thus, miR-206 and other muscle-specific miRNAs in serum are useful for monitoring the DMD pathological progression, and hence as potential non-invasive biomarkers for the disease.

There has been a long-standing need for reliable, non-invasive biomarkers for Duchenne muscular dystrophy (DMD). We found that the levels of muscle-specific microRNAs, especially miR-206, in the serum of DMD were 2- to 4-fold higher than in the controls. High levels corresponded to low muscle strength, muscle function, and quality of life (QoL). These miRNAs were able to discriminate DMD from controls by receiver operating characteristic (ROC) curves analyses. Thus, miR-206 and other muscle-specific miRNAs are useful as non-invasive biomarkers for DMD.

Abbreviations used
AUC

area under ROC curve

CK

creatine kinase

DMD

Duchenne muscular dystrophy

miRNAs

microRNAs

MRC

Medical Research Council

PCR

polymerase chain reaction

PedsQLTM

pediatric quality of life inventoryTM

QoL

quality of life

qRT-PCR

real-time quantitative RT-PCR

ROC

receiver operating characteristic

RT

reverse transcription

Duchenne muscular dystrophy (DMD) is a lethal X-linked disease that affects 1 : 3500 live male births (Emery 1991). The specific molecular defect is an absence or marked deficiency of dystrophin, a large membrane-associated protein that is part of the dystrophin–glycoprotein complex (Kunkel et al. 1986). Affected children will progressively lose muscle function which results in loss of independent ambulation between ages 7 and 12 years, and death usually occurs in their 20th year (Brooke et al. 1989).

Creatine kinase (CK) is an enzyme related to energy metabolism that is present in various types of cells (Ventura-Clapier et al. 1998). CK is commonly used as a blood-based biomarker for DMD to evaluate the level of muscle damage and necrosis and the efficacy of potential therapies, it is markedly increased in the early stages of the disease (10–100 folds higher than healthy controls) (Urganci et al. 2006). But CK is not always a reliable biomarker because it does not correlate with clinical assessments, and is influenced by the age and physical activity of the child (Zatz et al. 1991; Malm et al. 2000; Kim et al. 2010). Other biomarkers for DMD, such as lactate dehydrogenase, myoglobin, or aldolase also have the same problems (Percy et al. 1982; Kawai et al. 1991). Therefore, there has been a desire for more reliable, non-invasive biomarkers for DMD for a long time.

MicroRNAs (miRNAs) are small (~ 22 nucleotides), non-coding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level (Chen and Rajewsky 2007). The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species (Chen et al. 2008). Recently, miRNAs in serum are being seen as potential biomarkers for diseases such as cancers, liver injury, and heart failure (Mitchell et al. 2008; Starkey Lewis et al. 2011; Gidlof et al. 2013). These studies show that the levels of specific circulating miRNAs are associated with the development of these pathological processes. Certain miRNAs have tissue- or disease-specific expression profiles (Landgraf et al. 2007). Three families of muscle-specific miRNAs have emerged that are of importance in skeletal muscle development and function: miR-1-1/-1-2, -133a-1/-133a-2/-133b, and -206 (Sibley and Wood 2011). MiR-133 promotes proliferation of myoblasts by targeting serum response factor which has previously been shown to be an important transcription factor that negatively regulates muscle proliferation and promotes differentiation (Li et al. 2005). In contrast, miR-1 and -206 promote myogenesis through targeting among others, histone deacetylase 4 (HDAC4) (Chen et al. 2006) and connexin-43 (Cx43) (Kim et al. 2006). Muscle-specific miRNAs (miR-1, miR-133a, and miR-206) role as potential blood-based biomarkers has been demonstrated recently in the dystrophin-deficient muscular dystrophy animal models (Mizuno et al. 2011).

We hypothesized that the levels of muscle-specific miRNAs (miR-1, -133, and -206) in serum may be useful for monitoring the DMD pathological progression and therefore explored the possibility of these muscle-specific miRNAs as potential non-invasive biomarkers in children with DMD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

Subjects

Children with DMD and the controls of a similar age range were recruited between December 2010 and December 2012, in the department of neurology and the child health center of Children's Hospital, Chongqing Medical University, China. Table 1 summarizes the clinical characteristics of the subjects. Children with DMD inclusion criteria were as follows: (i) the diagnosis of DMD (Bushby et al. 2010) was initially based on their clinical history and neuromuscular findings and was confirmed by dystrophin gene testing or muscular biopsy, (ii) aged 4–12 years, (iii) still ambulant without any help, (iv) no severe or moderate learning difficulties or behavioral problems, (v) corticosteroid naïve. Exclusion criteria were as follows: (i) female DMD carrier status, (ii) families that did not agree to participate. Thirty-nine boys with DMD and 36 healthy subjects were included, and informed consent forms were signed by their parents. The study was approved in compliance with the Helsinki Declaration by the Ethics Committee of Children's Hospital, Chongqing Medical University, China.

Table 1. Clinical characteristics of the children with Duchenne muscular dystrophy (DMD) and the controls
Clinical characteristicsDMD (n = 39)Control (n = 36)p value
  1. Data are presented as mean ± SD or number.

  2. Statistical significance of the age difference between DMD and controls was assessed by two-tailed t-test. < 0.05 was considered statistically significant.

Age(year)
Mean ± SD7.88 ± 1.867.79 ± 2.120.99
Sex
Male3936 
Ambulant without any help3936 
No learning difficulties or behavioral problems3936 
Naïve corticosteroids3936 

Clinical evaluation

The clinical evaluations consisted of the following measurements: muscle strength – lower limb muscle groups were graded according to a grading system from the Medical Research Council (MRC) that had been expanded to a 10-point scale (Brooke et al. 1983; Florence et al. 1984). Timed functional testing tests included measurement of the time taken to walk 10 meters, to climb four standard steps, and to get up from supine position on the floor (Gowers' time) (Brooke et al. 1983; Florence et al. 1984). All timed activities were measured in seconds. Quality of life (QoL) questionnaire – the Chinese version of PedsQL™ 3.0 Neuromuscular Module was administered to children with DMD (Hu et al. 2013). Items are rated on a 5-point and transformed linearly to a 0–100 scale. Scale scores are computed as the sum of the items divided by the number of items answered. The order of testing, interval between tests, and procedures were standardized for each subject.

Sample collection and CK analyses

Approximately 2 mL of blood was collected from all of the subjects via a direct venous puncture into serum separator tubes, and serum CK levels were measured by Roche/Hitachi Modular Analytics Clinical Chemistry P 800 Module (Roche Diagnostics Corp., Indianapolis, IN, USA). Data were expressed as units per liter (U/L). To separate the serum for mRNA quantification, additional aliquots of blood were collected and centrifuged at approximately 3000 g for 10 min at 4°C. The resultant serum was dispensed into a 1.5 mL cryotube and stored at −80°C until further use.

RNA isolation and muscle-specific miRNAs quantification

Total RNA, including muscle-specific miRNAs, was extracted from 300-μL serum using TRIzol® reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instructions, and finally was dissolved in 40 μL RNase-free water. The concentration and purity of the small RNAs were measured with an ultraviolet spectrophotometer (Eppendorf Corp., Hamburg, Germany). The ratio of 260/280 was 1.7–2.0.

Total RNA was polyadenylated by Poly (A) Polymerase (NEB, Ipswich, MA, USA) and reverse transcribed using the Rever Tra Ace® miRNA Reverse Transcription kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The reverse transcription (RT) primers sequences used in the study are summarized in Table S1.

The SYBR Green® PCR Master Mix Kit (Toyobo) was used in real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) for relative quantification of miR-1, -133, and -206 in our study with U6 as an internal control (Ai et al. 2010; Zhang et al. 2010). The 20 μL of reaction solutions included 2 μL Diluted cDNA templates (1 : 5), 0.5-μL reverse primer, 0.5-μL sense primer, 10 μL of 2 × SYBR Green Master Mix, and final volume with RNase-free water. Table S1 summarizes the PCR primers sequences used in the study. qRT-PCR amplification was performed on 7500 FAST RT-PCR System (Applied Biosystems, Foster, CA, USA) according to the manufacturer's instructions. The reactions were incubated in a 96-well optical plate at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s, and then 72°C for 10 min. No signal was detected in negative control (no reverse transcriptase or no template). PCR was performed in triplicate for each sample for both these miRNAs and U6 at the same time. To validate the accuracy and specificity of the expected PCR product, we performed melting curve analyses (Figure S1).

The expression levels of muscle-specific miRNAs were normalized to those for U6 RNA. Relative miRNA production was determined with the 2−ΔΔCt method, where Ct is the threshold cycle. Differences in miR-1, -133, and -206 contents in the children with DMD compared with the controls were expressed as -fold changes (Livak and Schmittgen 2001).

Statistical analysis

Statistical analyses were performed with SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Continuous variables are presented as mean and standard deviation (mean ± SD). Statistical significance of differences between distributions was assessed by two-tailed t-test. Pearson's correlation analysis was used to examine correlation relationships. Receiver operating characteristic (ROC) curves and the area under ROC curve (AUC) were used to assess the feasibility of using miR-1, -133, and -206 in serum as a potential diagnostic tool for discriminating patients from normal controls (Soreide 2009). We used the Youden index for identification of the optimal cut-off point. All p values were two-sided, and < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

Muscle-specific miRNAs and CK levels in the serum of children with DMD

To explore the contents of muscle-specific miRNAs in the serum of children with DMD, we quantified the expression levels of miR-1, -133, and -206 in the serum of 39 boys with DMD by qRT-PCR, and compared those of 36 healthy control individuals. For miR-133, a single primer recognizing both miR-133a and miR-133b isoforms was utilized (Cacchiarelli et al. 2011a,b). The expression levels of miR-1, -133, and -206 were higher in the serum of boys with DMD (miR-1, 2.67 ± 1.14; miR-133, 2.16 ± 0.80; miR-206, 3.20 ± 1.20) than those in the controls (miR-1, 1.13 ± 0.29; miR-133, 1.15 ± 0.34; miR-206, 1.15 ± 0.30) (all < 0.01, Fig. 1).

image

Figure 1. Comparison of the levels of muscle-specific microRNAs (miR-1, -133, and -206) in serum of children with Duchenne muscular dystrophy (DMD) and the controls (39 DMD and 36 control cases) by quantitative reverse transcription-PCR (qRT-PCR). Values shown were normalized to U6. Differences between the levels were expressed as -fold changes with the 2−ΔΔCt method. The expression levels of miR-1, -133, and -206 were 2.67 ± 1.14, 2.16 ± 0.80, and 3.20 ± 1.20 in serum of children with DMD and those were 1.13 ± 0.29, 1.15 ± 0.34, and 1.15 ± 0.30 of controls. Data expressed as mean ± SD. All p values were two sided, and p < 0.05 was considered statistically significant. **p < 0.01 compared with the controls. miR, microRNA; SD, standard deviation.

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Muscle-specific miRNAs in the serum correlated with clinical evaluations

To study whether there were correlations between muscle-specific miRNAs and the clinical characteristics of children with DMD, Pearson's correlation analyses between the miRNAs levels and muscle strength, muscle function, and QoL of PedsQL™ 3.0 Neuromuscular Module were performed at the time of the molecular diagnosis. The results showed that miR-1, -133, and -206 expressions had nothing to do with age (> 0.05), but there were significant inverse correlations, especially in the case of miR-206, with muscle strength (miR-1, = −0.34, −0.40; miR-206, = −0.43), muscle function (miR-1, = 0.29, 0.33; miR-133, = 0.35, 0.29; miR-206, = 0.44, 0.39), and QoL (miR-1, = −0.34, −0.35; miR-133, = −0.50, −0.56; miR-206, = −0.51, −0.48) (all < 0.05). At the same time the CK level had a significant inverse correlation with age (= −0.45, < 0.05), but there was no relationship with muscle strength, muscle function, and QoL (> 0.05) (Table 2). Table 3 summarizes the results of the clinical evaluations and CK levels of the boys with DMD.

Table 2. Muscle-specific microRNAs (miRNA) and creatine kinase (CK) levels in serum of children with Duchenne muscular dystrophy (DMD) correlated with clinical evaluations
  n miR-1miR-133miR-206CK level
r p r p r p r p
  1. Pearson's correlation analyses between muscle-specific miRNAs/CK levels and clinical evaluations were performed. The clinical evaluations consisted of the following measurements: muscle strength – lower limb muscle groups were graded according to a grading system from the Medical Research Council (MRC) that had been expanded to a 10-point scale. Timed functional testing tests included measurement of the time to walk 10 meters, to climb four standard steps, and to get up from supine position on the floor (Gowers' time). All timed activities were measured in seconds. Quality of life (QoL) questionnaire – the Chinese version of PedsQL™ 3.0 Neuromuscular Module was administered to children with DMD. Items are rated on a 5-point and transformed linearly to a 0 to 100 scale. Scale scores are computed as the sum of the items divided by the number of items answered. All p values were two sided, and < 0.05 was considered statistically significant.

Age39−0.170.32−0.220.21−0.270.12−0.450.01
Lower limb muscle strength
Hip flexion: R39−0.340.04−0.210.22−0.430.010.030.85
Knee extension: R39−0.400.02−0.230.19−0.430.010.090.61
Timed functional testing
Time to walk 10 m390.290.080.350.040.440.01−0.170.32
Time to climb 4 steps360.330.050.240.160.390.02−0.190.27
Time for Gowers' sign test340.110.530.290.090.130.45−0.070.70
PedsQL™ 3.0 NMM (total scale)
Child self-report23−0.340.04−0.50< 0.01−0.51< 0.01−0.150.37
Parent proxy-report39−0.350.04−0.56< 0.01−0.48< 0.01−0.090.62
Table 3. The results of the clinical evaluations and creatine kinase (CK) level of children with Duchenne muscular dystrophy (DMD)
Clinical evaluations/CK level n Results
  1. The clinical evaluations consisted of the following measurements: muscle strength – lower limb muscle groups were graded according to a grading system from the Medical Research Council (MRC) that had been expanded to a 10-point scale. Timed functional testing tests included measurement of the time to walk 10 meters, to climb four standard steps, and to get up from supine position on the floor (Gowers' time). All timed activities were measured in seconds. Quality of life (QoL) questionnaire – the Chinese version of PedsQL™ 3.0 Neuromuscular Module was administered to children with DMD. Items are rated on a 5-point and transformed linearly to a 0–100 scale. Scale scores are computed as the sum of the items divided by the number of items answered.

Lower limb muscle strength (score)
Hip flexion: right396.89 ± 1.26
Knee extension: right397.44 ± 1.21
Timed functional testing (second)
Time to walk 10 m3911.08 ± 2.60
Time to climb 4 steps367.64 ± 4.03
Time for Gowers' sign test347.95 ± 4.05
PedsQL™ 3.0 Neuromuscular Module (total scale, score)
Child self-report2356.51 ± 10.26
Parent proxy-report3955.86 ± 9.41
CK level (U/L)3911617.77 ± 5640.21

Three of the boys with DMD could not do the test to climb four steps and four of them could not do the Gowers' test, and so they were excluded from the statistics of the timed functional testing. Sixteen of the boys with DMD did not complete the child self-assessment survey PedsQL™ 3.0 Neuromuscular Module, because they were less than 7 years old (Table 2, 3).

Evaluation of muscle-specific miRNAs in serum as potential diagnostic markers

To evaluate whether muscle-specific miRNAs in serum can be used as potential diagnostic markers for DMD, ROC curve analyses were obtained by plotting the rate of true positive (sensitivity) vs. false positive (1-specificity). These results revealed that miR-1, -133, and -206 were able to discriminate DMD from healthy controls with AUC of 0.93, 0.90, and 0.96 separately. At the cut-off values of 0.78, 0.68, and 0.90, the sensitivities and specificities of miR-1, miR-133, and miR-206 were 83% and 95%, 78% and 91%, 94% and 95%, respectively (Fig. 2).

image

Figure 2. Receiver operator characteristic (ROC) curves of the muscle-specific miRNAs (miR-1, -133, and -206) regarding the diagnostic power of these markers to distinguish healthy from dystrophic cases. Areas under ROC curve (AUC) values are also shown. AUC of miR-1, -133, and -206 were 0.93, 0.90, and 0.96 separately. At the cut-off values of 0.78, 0.68, and 0.90, the sensitivities and specificities of miR-1, miR-133, and miR-206 were 83 and 95%, 78 and 91%, 94 and 95%, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

Recently, several studies have reported that miRNAs in serum are promising biomarkers for diseases, such as cancers, liver injury, and heart failure (Mitchell et al. 2008; Starkey Lewis et al. 2011; Gidlof et al. 2013). The discovery of muscle-specific miRNAs (miR-1, -133, and -206) in the serum of children with DMD could open up new possibilities of potential non-invasive biomarkers for muscular dystrophy. We demonstrated that muscle-specific miRNAs, especially miR-206, were highly abundant in the bloodstream of the boys with DMD (up to 3.20 ± 1.20, 2−ΔΔCt): almost 2- to 4-fold enriched in comparison to samples from the healthy controls (less than 1.15 ± 0.34, 2−ΔΔCt). The surprising increase of muscle-specific miRNAs in serum was explained by the observation that miRNAs could be part of exosomal particles (Mitchell et al. 2008), or caused by an increase in leakage or secretion of miRNAs from muscle, and not by any change in expression in skeletal muscle (Mizuno et al. 2011).

The existing biochemical markers for DMD include CK, lactate dehydrogenase, myoglobin, and aldolase (Percy et al. 1982; Kawai et al. 1991; Urganci et al. 2006). Among the aforementioned markers, CK has been considered one of the most common markers. But it can only detect 50–70% of the carriers of DMD (Dreyfus et al. 1966; Percy et al. 1982; Schapira et al. 1987), is not always reliable in reflecting the pathological condition of muscular diseases, and is influenced by the age and physical activity of the child (Malm et al. 2000; Zatz et al. 1991; Davies 1997). Interestingly, when comparing the levels of miR-1, -133, and -206 in the serum with the functional performances of children with DMD, we observed a very good inverse correlation: high levels of muscle-specific miRNAs corresponded to low muscle strength, muscle function, and QoL. Notably, quantifying muscle-specific miRNAs are better potential markers than CK activity, and they display fluctuations more related to the pathological condition of the disease (Cacchiarelli et al. 2011a,b; Mizuno et al. 2011), little influenced by age and stress (Mizuno et al. 2011), and they are stable, lasting for a long time in serum (Chen et al. 2008; Mitchell et al. 2008). Our analyses were performed on children with DMD who could ambulate (4–12 years) as a very well-established and reliable clinical assessment for the quantification of disease severity is available from MRC guidelines (Brooke et al. 1983; Florence et al. 1984). At the same time, a QoL questionnaire was administered by the Chinese version of the PedsQL™ 3.0 Neuromuscular Module, which has acceptable psychometric properties and it is a reliable measure of disease-specific health-related quality of life (HRQOL) in Chinese children with DMD (Hu et al. 2013).

ROC curve analysis is a useful tool in the assessment of biomarker accuracy in two situations – acknowledging strengths and weaknesses of the method (Soreide 2009). In this study, the ROC curves revealed that miR-1, -133, and -206 were able to discriminate DMD from healthy controls with AUC ≥ 0.90, and with high sensitivity and specificity. The results further confirmed the superiority of muscle-specific miRNAs, especially miR-206, in serum as reliable and promising biomarkers of DMD.

As well as potential non-invasive biomarkers for the diagnosis of DMD, muscle-specific miRNAs can also be used as biomarkers for monitoring the outcomes of therapeutic interventions as shown in the mdx mouse model (Cacchiarelli et al. 2011a,b). In human DMD myoblasts treated with exon skipping, it was found that interfering with miR-31 activity at the same time increases dystrophin rescue and can provide an ameliorating strategy for DMD therapy (Cacchiarelli et al. 2011a,b). At the same time, miR-206 in activated satellite cells regulates the transition from proliferation to differentiation during skeletal muscle regeneration, which slows progression of DMD in mice (Chen et al. 2010; Liu et al. 2012). In addition, miR-206 delays disease progression and promotes regeneration of neuromuscular synapses in amyotrophic lateral sclerosis model mice (Williams et al. 2009). Taken together, these results indicate that muscle-specific miRNAs are new and valuable biomarkers for the diagnosis of DMD, as well as possibly monitoring the outcomes of therapeutic interventions and also as a novel therapeutic approach in humans in the future.

The limitations of our study were that our sample size was relatively small, and we did not collect the long-term follow-up data. The relationship of muscle-specific miRNAs with clinical characteristics should be explored in more detail by conducting further studies with non-ambulant patients. At the same time, muscle-specific miRNAs could be used as biomarkers for monitoring the outcomes of corticosteroid therapeutic interventions in humans. Importantly, muscle-specific miRNAs delay DMD disease progression in mice, which opens avenues to their possible therapeutic application in human muscular diseases. This is an area that clearly needs further research.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

In this report, we focused on muscle-specific miRNAs (miR-1, -133, and -206) and found that they are significantly increased in the serum of children with DMD. High levels of muscle-specific miRNAs, especially miR-206, correspond to low muscle strength, muscle function, and QoL. Our studies indicate that the quantification of muscle-specific miRNAs, or even just one of them, in serum, can be used as potential non-invasive biomarkers to reveal DMD conditions in humans, and possibly for monitoring the outcomes of therapeutic interventions and also as a novel therapeutic approach in humans in the future.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

We sincerely thank the children and parents who took part in this study. We are also grateful to all the experts involved in the study and support from the Children's Hospital, Chongqing Medical University. This work was funded by Research Project of Chongqing Municipal Health Bureau (No. 2012-1-044) and Youth Research Project of Fujian Provincial Health Bureau (No. 2009-2-22).

Informed consent was achieved for all subjects, and the experiments were approved by the local ethics committee. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

Authors' contributions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information

JH designed experiments, set up and performed RNA procedures and qRT-PCR, and wrote the manuscript; MK and SH collected human sera and performed clinical assessments; YY and LC performed qRT-PCR and statistical analyses; LJ conceptualized and designed the study, supervised the data analysis, and revised the manuscript. All authors read and approved the final manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments and conflict of interest disclosure
  8. Authors' contributions
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
jnc12662-sup-0001-FigS1-TableS1.pdfapplication/PDF101K

Figure S1. Melting curve analyses of qRT-PCR reaction of each miRNA.

Table S1. Reverse transcription and polymerase chain reaction primers used in the study.

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