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Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients
PNP-08773; No of Pages 7 Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
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Article history: Received 5 December 2014 Received in revised form 8 May 2015 Accepted 11 May 2015 Available online xxxx
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Keywords: Biomarker microRNA miR-132 Peripheral blood Schizophrenia
School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, PR China Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine in Henan Province, Xinxiang 453003, PR China The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang 453002, PR China d School of Pharmacy, Xinxiang Medical University, Xinxiang 453003, PR China e Department of Psychology, Xinxiang Medical University, Xinxiang 453003, PR China f Department of Pharmacology, School of Medicine, Zhengzhou University, Zhengzhou 450001, PR China
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Hai-chuan Yu a,b,c,f, Jiao Wu d, Hong-xing Zhang e,c, Gao-li Zhang a, Juan Sui a, Wen-wen Tong a, Xin-Ya Zhang a, Li-li Nie c, Ju-hong Duan a, Li-rong Zhang f, Lu-xian Lv c,⁎ c
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Alterations in microRNAs (miRNAs) have been considered to have diagnostic implications in most diseases, but few studies have reported dysregulated miRNAs in schizophrenia (SCZ). In order to observe an association between miRNAs and SCZ, this study was designed to investigate expression profiling of miRNAs in peripheral blood mononuclear cells (PBMCs). miRNA microarray technology was employed to compare the expression of miRNAs in PBMCs from SCZ patients (n = 105) and normal controls (n = 130), and real-time quantitative polymerase chain reaction (QPCR) was used to analyze the results. Several important miRNA levels were examined before and after antipsychotic treatment in first-onset SCZ patients. In addition, an SCZ-like rat model was established using dizocilpine (MK-801), and miR-132 expression in PBMCs and whole-brain tissue from SCZlike rats was studied using QPCR. In humans, dysregulated miRNAs were observed before treatment and QPCR verified that miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 levels were significantly decreased (P b 0.01 for all) in PBMCs of SCZ patients compared with healthy controls. After antipsychotic treatment there was a marked increase in miR-132 (P b 0.01), miR-664⁎ (P b 0.05) and miR-1271 (P b 0.05) levels in SCZ patients compared with the levels before treatment. In the animal assays, miR-132 levels declined in PBMCs and wholebrain tissues (both P b 0.05) of the SCZ-like rats compared to controls. For the first time, our results suggest that miR-132 is a potential and superior biomarker in peripheral blood that will allow discrimination of SCZ patients from healthy controls. © 2015 Published by Elsevier Inc.
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development, failure to develop appropriate neuronal phenotype, neuronal atrophy, severe growth defects and early death (Schaefer et al., 2010). miR-133b regulates the maturation and function of midbrain dopamine neurons through a negative feedback circuit involving the transcription factor pituitary homeobox 3 (Pitx3) (Tognini et al., 2011). miR-124, which promotes neuronal gene expression in differentiating neural progenitor cells, is enriched in mammalian brain. There is a feedback regulatory loop between miR-9, a brain-specific miRNA, and the nuclear receptor TLX, which plays an important role in maintaining neural stem cells in the proliferative and self-renewable state during neural stem cell differentiation (Zhao et al., 2009). miR-132 and miR-134 may functionally control synaptic plasticity, and transcription factor myocyte enhancing factor 2 (Mef2) increases miR-134 expression in response to neuronal activity (Fiore et al., 2009). All of these studies have revealed dysfunction in miRNA signaling contributes to neurodegenerative and psychiatric disorders. Schizophrenia (SCZ) is a severe psychiatric disorder that has an average life time development risk of 0.7%; heritability of the disease is
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Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients
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1. Introduction
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MicroRNAs (miRNAs) are 20–25-nucleotide, single-stranded, noncoding RNA molecules that play a pivotal role in diverse cellular processes including cell-cycle control, immunity, viral or bacterial disease, stem-cell differentiation, development, metabolism and oncogenesis (Cech and Steitz, 2014; Lujambio and Lowe, 2012; Wu et al., 2014). Accumulating evidence supports a key role for miRNAs in neurogenesis, neuronal maturation and brain development (Belzeaux et al., 2012; Im and Kenny, 2012; Kapsimali et al., 2007; Sun and Shi, 2014a). In zebrafish, deletion of Dicer results in defects in neural development, and injection of miR-430 can rescue many of these defects (Giraldez et al., 2005). Dicer-deficient mice display marked deficits in brain
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⁎ Corresponding author at: Department of Psychiatry of the Second Affiliated Hospital of Xinxiang Medical University, No.388, Jianshe Middle Road, Xinxiang 453002, PR China. Tel.: +86 373 3373969. E-mail address: [email protected] (L. Lv).
http://dx.doi.org/10.1016/j.pnpbp.2015.05.007 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xinxiang Medical University, and written informed consent was obtained from all participants. Between August 2011 and June 2014, 105 patients with SCZ (50 males and 55 females; mean age: 25.03 ± 8.34 years) and 130 healthy controls (60 males and 70 females; mean age: 22.73 ± 6.79 years) were recruited. Individuals with a history of severe medical complications, organic brain disease, any concomitant major psychiatric disorders, or substance dependence were excluded. The consensus diagnoses for SCZ patients were made by at least two psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition IV (DSM-IV). The clinical features of 105 patients were evaluated by the senior psychiatrist using Positive and Negative Symptom Scale (PANSS). During the same period the first-onset schizophrenia patients, who were Chinese of Han descent, were selected and treated with risperidone initially. According to the principle of treatment in our study, patients who experienced an increase in body weight of more than 10%, serious side effects, or no significant improvement in 8 weeks were
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The peripheral blood samples (5 mL per participant) were collected in EDTA anticoagulant tubes and mononuclear cells were separated by density gradient centrifugation using a percoll density gradient (d = 1.077; Amersham Pharmacia Biotech, Germany). Total RNA was extracted from PBMCs with Trizol reagent (Invitrogen, CA, USA) following the manufacturer's instructions and stored at −80 °C until use. For animal assays, blood samples and whole brain tissues were obtained and RNA was isolated in the same manner as above. Total RNA was extracted from different PBMC samples using TRIzol reagent (Invitrogen) and quantified respectively. DNA was removed by treatment with 5 units of DNase I (Promega, Madison, WI) at 37 °C for 45 min followed by inactivation at 65 °C for 10 min. Then firststrand of cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instruction. Stemloop reverse transcription and QPCR for miRNAs were executed as described previously (Chen et al., 2005). cDNA was synthesized from 2 μg of total RNA using reverse transcribed primers as shown in Supplementary Table 1.
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excluded. In addition, some patients were lost to follow-up because of low compliance or noncooperation. After screening, only 10 patients maintained and fulfilled the monotherapy regimen and achieved remission. PANSS was used to evaluate the severity of SCZ and clinical efficacy of treatment. Risperidone was given once daily, with an initial dose of 2 mg/day, increasing up to 3–6 mg/day over the following weeks. The maintenance dose was adjusted according to side effects and clinical assessment. All patients received oral risperidone treatment after baseline assessments and achieved remission after 8 weeks of treatment.
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estimated at 80% based on twin studies (Sullivan et al., 2003; Wright et al., 2013). Although the pathogenetic mechanisms leading to SCZ are unclear, it is well accepted that SCZ is the result of a complex interplay between genetic variants and environmental factors. Recently, there has been much interest in the possible involvement of miRNAs in SCZ (Im and Kenny, 2012; Sun and Shi, 2014a). The findings of the largest Genome-Wide Association Study (GWAS) on SCZ have revealed that the strongest association of SNP with SCZ lies within the intron of a putative primary transcript for miR-137 (Hill et al., 2014; Schizophrenia Psychiatric Genome-Wide Association Study, 2011). Xu et al. (2010) have indicated that miR-30e ss178077483 plays a role in SCZ susceptibility. In animal studies, Kocerha et al. (2009) have found that miR219 mediates the behavioral effects of dizocilpine (MK-801) treatment in mice. An in situ hybridization study has revealed miR-185 expression in brain regions is implicated in SCZ mice, and gene-based tests revealed that converse expression patterns for miR-185 and its target genes (ATAT1, SH3PXD2A, NTRK3) provided no further evidence of their involvement in SCZ. miR-181b, miR-30e, miR-34a and miR-7 are probably involved in the pathogenesis of SCZ, and significant downregulation of miR-181b expression has been observed after treatment with atypical antipsychotics as an antipsychotic response (Song et al., 2014). Miller et al. (2012) have suggested that miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes, DNMT3A, GATA2 and DPYSL3, contribute to the neurodevelopmental and neuromorphological pathologies present in SCZ. Despite growing evidence showing a relationship between altered expression of miRNAs and SCZ, it is unclear which miRNA has the greatest influence upon SCZ and which miRNA can be considered a diagnostic biomarker. In this study, we tried to identify miRNAs that are clinically practicable biomarkers for SCZ through blood-based detection. We compared the miRNA expression profiles of peripheral blood mononuclear cells (PBMCs) by using miRNA microarray technology and real-time quantitative polymerase chain reaction (QPCR), and several markedly different expression levels of miRNAs were observed. Of importance, downregulation of miR-132 in PBMCs stands out in accordance with Miller's results (Miller and Zeier, 2012), which showed that miR-132 was significantly decreased in the prefrontal cortex of SCZ patients. In addition, miR-132 was involved in the pathogenesis of SCZ and significant upregulation of miR-132 expression was found in SCZ patients' PBMCs after antipsychotic treatment. Animal studies demonstrated miR-132 downregulation is associated with SCZ-like animals after MK-801 treatment. All of these results proved that miR-132 may be a potential biomarker in peripheral blood for the diagnosis of SCZ.
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2.3. miRNA expression profiling
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Sixty total RNA samples from PBMCs of SCZ patients and 60 total RNA samples from normal controls were selected and pooled to 6 case pools and 6 control pools (every 10 samples RNA were mixed equivalently in one tube as one pool), respectively. Global miRNA expression profiling of SCZ patients and normal controls was investigated using miRNA microarray (Agilent Human miRNA V19.0) that contained capture probes for 2006 human miRNAs that were registered and annotated in the Sanger miRBase V19.0.
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2.4. QPCR analysis for miRNAs
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QPCR was carried out using the SYBR Premix Ex Taq kit (Takara, Dalian, China) according to the manufacturer's instructions using QPCR primers (Supplementary Table 1). U6 snRNA was used as the endogenous control and the comparative Ct method was used to quantify target genes relative to their endogenous control. Each reaction was performed in triplicate. For each individual analysis, one of the samples (from one healthy subject) was designated as the calibrator and given a relative value of 1.0. All quantities were expressed as n-fold relative to the calibrator.
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2.5. Northern blot analysis for miR-132
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Northern blot analysis of miRNAs was performed as described previously with minor modifications (Wang et al., 2011). Total RNA was subjected to 15% TBE–Urea gels and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Germany) and crosslinked by ultraviolet irradiation. A γ-32P end-labeled miRNA probe of miR-132 was employed, and U6 snRNA was used as a control probe to verify equal RNA loading.
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Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Quantified microarray signal intensity values were normalized to per-chip mean values. Differentially expressed miRNAs were identified using the two-class paired t test procedure within significant analysis of microarrays (SAM) with a 5% false discovery rate (FDR) threshold. Cluster and Java TreeView were used to build the unsupervised tree. The genes and arrays were mean-centered, and hierarchical trees were built using correlation metrics. Statistical analyses of QPCR data were performed with the GraphPad Prism 6 and SPSS version 21.0 (SPSS, Chicago, IL). For each pair of sample types, a two-sample t test assuming equal variance was carried out for miRNAs. We used a conventional receiver-operating characteristic (ROC) curve to analyze miRNA levels and to determine the cutoff points that yielded sensitivity and specificity. We calculated the area under the curve (AUC) and 95% confidence intervals for the AUC. The association with statistical significance was assessed by nonparametric tests.
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3.1. Baseline data
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In our study, all patients in the study group suffered from SCZ. Their age, gender, smoking history, ethnicity and educational level were recorded, and the clinical characteristics and demographics of the SCZ and control groups are shown in Supplementary Table 2. No significant differences were found between the SCZ patients and healthy controls in age (t = 0.023, P = 0.87) or sex (χ2 = 0.104, P = 0.84). At the same time, to investigate the changes in expression of miRNAs before and after antipsychotic therapy, the first-onset SCZ patients (n = 12) were selected and treated with risperidone initially.
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3.2. Analysis of miRNA microarray
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To investigate the potential involvement of miRNAs in SCZ, we scanned global miRNA expression profiles of PBMCs from 60 SCZ patients (6 pools) and 60 healthy controls (6 pools) using miRNA microarray for mature human miRNAs. Following comparison between the SCZ patients and healthy controls, we identified certain miRNAs, the majority of which showed decreased expression in SCZ patients. Among them, we focused on 41 downregulated miRNAs (Fig. 1), whose expressions were significantly decreased in SCZ patients, and the change folds were all greater than 2.5 compared with controls (Supplementary Table 3).
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3.3. QPCR validation of the results from miRNA microarray
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To verify the accuracy and universality of the miRNA microarray, QPCR analysis was performed on the differentially expressed miRNAs of PBMCs in expanded samples, including 105 SCZ patients and 130
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Male Sprague-Dawley rats (200–250 g) were provided by the Experimental Animals Center of Xinxiang Medical University. All experimental protocols were reviewed and approved by the Animal Care and Use Committee of Xinxiang Medical University. Thirty SD rats were randomly assigned to two batches: a chronic MK-801-induced SCZ-like rat model (n = 15) was established by repeated intraperitoneal administration of MK-801 (dissolved in saline, 0.3 mg/kg/day), and rats that received saline once a day for 4 weeks served as controls (n = 15). After 4-week administration of MK-801, an open field test (total distance traveled, cm) and social interaction assay were performed as described previously (Guo et al., 2010; Huang et al., 2014). Rats were injected with MK-801 or saline 30 min prior to behavioral tests and were put into an open arena and measured.
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Fig. 1. Differential expression of miRNAs in SCZ patients and healthy controls. Unsupervised hierarchical clustering analysis of miRNA expression profiles in 6 pools from PBMCs of SCZ patients and 6 pools from healthy controls. The degree of relatedness of the expression patterns in the samples is represented by the dendrogram at the left of the panel. Each column corresponds to the expression profile of a sample, and each row corresponds to a miRNA. The color in each cell reflects the level of expression of the corresponding miRNA in the corresponding sample. Increasing intensities of red mean that a specific miRNA has a higher expression in the given sample and increasing intensities of green mean that this miRNA has a lower expression. The scale reflects the miRNA abundance ratio in a given sample relative to the mean level for all samples. The red arrow presents the focus miRNAs of this paper. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
healthy controls. Average expression levels of individual tested miRNAs from SCZ patients and healthy controls are shown in Supplementary Fig. 1; expression levels of all selected miRNAs of SCZ patients were compared with those of healthy controls. From these QPCR results, a significant decrease (P b 0.01) in miRNA level was noted in PBMCs of SCZ patients for six miRNAs (miR-132, miR-664⁎, miR-1271, miR-200c, miR-432, miR-134). One of the normal healthy samples (N-1) was designated as the calibrator and the relative expressions of other samples were calculated by comparing the individual expressions of all miRNAs with those of this calibrator. We measured the expression levels of the miRNAs for the SCZ patients and healthy controls, and found that the relative expression levels of miR-132 (median: SCZ 0.59, healthy controls 1.30, P b 0.01, Fig. 2A), miR-134 (median: SCZ 0.54, healthy controls 1.37, P b 0.01, Fig. 2B), miR-1271 (median: SCZ 0.67, healthy controls 1.66, P b 0.01, Fig. 2C), miR-664⁎ (median: SCZ 1.10, healthy controls 1.70, P b 0.01, Fig. 2G), miR-132 (median: SCZ 0.64, healthy controls 1.46, P b 0.01, Fig. 2H) and miR-432 (median: SCZ 0.62, healthy controls 1.2, P b 0.01, Fig. 2I) were significantly lower in SCZ patients than those in healthy controls. No significant differences in other miRNA levels were observed between SCZ patients and healthy control
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Fig. 2. Discrimination of miRNA levels in PBMCs of SCZ patients (n = 105) and healthy controls (n = 130). (A), (B), (C), (G), (H), and (I): miR-132, miR-134, miR-1271, miR-664⁎, miR200c, and miR-432 expressions, respectively, in PBMCs of SCZ patients and healthy controls. There were significant decreases in miR-132, miR-134, miR-1271, miR-664⁎, miR-200c, and miR-432 in PBMCs of SCZ patients (P b 0.01). (D), (E), (F), (J), (K), and (L): ROC curves were drawn for miR-132, miR-134, miR-1271, miR-664⁎, miR-200c, and miR-432, which yielded 0.934, 0.920, 0.921, 0.836, 0.878, and 0.821 as the AUC values, respectively.
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groups, indicating the uniqueness of miR-132, miR-664⁎, miR-1271, miR-200c, miR-432 and miR-134 as SCZ biomarkers. ROC analysis was subsequently used to assess the diagnostic sensitivity and the specificity (Fig. 2). Diagnostic sensitivities of miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 for SCZ were 94%, 92%, 91%, 88%, 93% and 88%, respectively. The corresponding specificities were 89%, 82%, 81%, 74%, 78% and 68%, and ROC curve areas (95% CI) were 0.934 (89.4–97.5%), 0.920 (88.0–96.0%), 0.921 (88.1–96.1%), 0.836 (78.0–89.1%), 0.878 (82.8–92.7%) and 0.821 (76.5–87.6%), respectively (Table 1). The data showed the significant diagnostic value of
miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 for 278 SCZ, and these miRNAs may be novel noninvasive biomarkers for the 279 diagnosis of SCZ. 280 3.4. miRNA expression differences before and after antipsychotic treatment 281 in first-onset SCZ patients 282 To investigate the changes in these miRNA levels before and after an- 283 tipsychotic treatment, blood samples were collected on the day before 284 (baseline) and the day after 8 weeks of risperidone treatment (end 285
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
H. Yu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2015) xxx–xxx
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Receiver-operating characteristic (ROC) curve analysis was used to determine the cutoff points that yielded the highest combined sensitivity and specificity. The relative expression of miRNA copies to U6 snRNA copies were used to perform the ROC curve analysis.
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point) and used for QPCR detection. Details are provided in Supplementary Table 4. Risperidone is one of the most widely used first-line antipsychotics for acute SCZ. We measured the expression levels of these six miRNAs in peripheral blood of SCZ patients before and after risperidone treatment, and the results are shown in Fig. 3. According to these data, the expression of miR-132 was significantly upregulated after risperidone treatment compared to before risperidone treatment (P b 0.01) (as in Fig. 3A), while the levels of miR-1271 and miR-664⁎ were also moderately higher (P b 0.05) after risperidone treatment (as in Fig. 3C and D), but there were no significant differences in the levels of miR-134, miR-200c and miR-432. These results indicated that risperidone improved certain miRNAs levels in SCZ patients significantly; in particular, miR-132 may potentially be involved in a therapeutic strategy as an effective biomarker.
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3.5. Changes in expression of miR-132 in the MK-801 SCZ-like rat model
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In animal assays, locomotor activity was recorded for 30 min as an indicator of “SCZ-like” behavior. MK-801 treated rats (n = 15) exhibited significantly increased locomotor activity compared with salinetreated controls (n = 15), as expected. At the same time, social interaction was tested through videotaping for 20 min and a decrease in social interaction duration was observed in the MK-801 group compared with the saline group (Supplement Fig. 2). Therefore, the MK-801 SCZ-like rat model was established successfully. QPCR and Northern blot results
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Multiple studies have shown that defective neuronal plasticity and function in neurodevelopmental disorders may have resulted from impaired posttranscriptional regulation mediated by miRNAs (Hunsberger et al., 2009) and that miRNAs play important roles in the etiology of neuropsychiatric and neurodevelopmental disorders, and neurodegenerative diseases (Olde Loohuis et al., 2012; Sun and Shi, 2014b). miR132 expression has been reported to be dysregulated in Alzheimer's disease and SCZ (Cogswell et al., 2008). Kim et al. (2010) have proved that miR-132 expression is correlated with SCZ by examining the postmortem prefrontal cortex of patients with SCZ. Miller and Zeier, (2012) have examined the expression of miRNAs in prefrontal cortical tissue from 100 control and schizophrenic subjects and found that miR-132 was significantly downregulated in SCZ patients compared with controls. Now, we have observed a significant decrease in miR132 in peripheral blood of SCZ patients, and these data are in accordance with Miller's results in prefrontal cortical tissue. The reduced levels of miR-132 in peripheral blood and prefrontal cortical tissue may be reflecting the same characteristic of SCZ, and all of these results illustrated that miR-132 is a potential target for diagnosis and treatment of diseases related to SCZ. Recent studies have suggested that aberrant expression of miRNAs is a potential diagnostic biomarker of disease (Jansen et al., 2014; Li et al., 2013; Li et al., 2014; Silva et al., 2014). Since brain tissue is not readily accessible, a new focus in research on biomarkers is blood-based expression profiling of miRNAs. For blood-based miRNA detection, PBMCs are more stable and feasible to use than serum because the contents of miRNAs in serum are very low. However, it has been unclear which miRNAs in human peripheral blood can be regarded as biomarkers for SCZ, and how the expressions of these miRNAs change before and after antipsychotic therapy. In this study, miRNA microarray was employed to identify the differences in miRNA expression in peripheral blood between SCZ patients and healthy controls, and 41
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indicated that the expression level of miR-132 was significantly lower in 309 PBMCs (Fig. 4A and C) and whole brain tissue (Fig. 4B and D) of the SCZ- 310 like rats than in the normal control group. 311
Table 1 Diagnostic accuracy based on PBMC miRNA levels.
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Fig. 3. (A–F) Comparison of miRNA levels in PBMCs before and after 8 weeks of risperidone treatment in first-onset SCZ patients (n = 10) using QPCR. Significant increases in miR-132 (P b 0.01), miR-1271 (P b 0.05), and miR-664⁎ (P b 0.05) were found after treatment when compared with before.
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Contributors
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Hai-chuan Yu, Lu-xian Lv, and Li-rong Zhang designed the study. Hai-chuan Yu and Jiao Wu wrote the first draft of the manuscript. Haichuan Yu, Wen-wen Tong, Gao-li Zhang, Gao-li Zhang, and Juan Sui performed the experimental work. Jiao Wu and Hong-xing Zhang undertook statistical analyses. Li-li Nie and Ju-hong Duan undertook all sample collections and support. All authors contributed to and have approved the final manuscript.
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(No. 13B350215, No. 14B320015), Foundation for Excellent Innovation Talents of Health Science and Technology by Henan Health Department (No. cxyx20130418) and Scientific Research Fund Project of Xinxiang Medical University (No. 100403, No. 505002).
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This work was financially supported by the National Natural Sciences Foundation of China (No. 31301135), Science and Technology Research Projects of the Education Department of Henan Province
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The authors wish to thank all the subjects who participated in the study. Authors also acknowledge invaluable assistance by numerous mental health professionals in the different clinical departments of the Second Affiliated Hospital of Xinxiang Medical University.
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Acknowledgments
References
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downregulated miRNAs were found. Then, QPCR was used to validate the data from miRNA microarray, and six miRNAs (miR-132, miR664⁎, miR-1271, miR-200c, miR-432 and miR-134) exhibited significant decreases (P b 0.01) in peripheral blood of 105 SCZ patients compared to 130 healthy controls. To investigate whether the six miRNAs may be informative as SCZ therapeutic markers, we compared miRNA expression in peripheral blood from first-onset SCZ patients before and after antipsychotic therapy. We found that the expressions of miR-132, miR-1271 and miR664⁎, but not miR-134, miR-200c and miR-432, increased in PBMCs after risperidone treatment compared to before treatment. In addition, the MK-801 SCZ-like model was established successfully and the six miRNAs were detected. QPCR and Northern blot analysis demonstrated that miR-132, but not the other miRNAs, was reduced in PBMCs or whole brain tissue of rats with MK-801 treatment compared to normal saline controls. Based on these findings, we suggest that miR-132 could be a superior marker for detection of SCZ by blood tests. Indeed, the miR-132 level decreased going from non-SCZ patients to SCZ patients and was markedly increased after risperidone treatment. In further study, the important target genes of miR-132 involved in SCZ will be investigated by using dual luciferase reporter assays and Western blot, and the expression changes of these genes in PBMCs from SCZ patients will be measured. In general terms, the identification of SCZ-specific miRNA profiles in PBMCs is an emerging field of particular interest, but it can be stated that the study of miRNAs in SCZ is in its early stages. Only a handful of very recent studies have shed a little light on this growing field. Although the role of miRNAs in the clinical management of psychiatric disease has yet to be studied, our work shows that they are exciting potential candidates for this challenge. Taken together, our observations have demonstrated that miR-132 plays an important role as a diagnostic biomarker in SCZ. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pnpbp.2015.05.007.
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Fig. 4. Compared with the normal group (n = 15), the expression level of miR-132 was significantly downregulated in PBMCs and whole brain tissue of SCZ-like rats (n = 15). (A) QPCR analysis of miR-132 expression in PBMCs. (B) QPCR analysis of miR-132 expression in whole brain tissue. (C) Northern blot analysis indicated a decrease in miR-132 level in PBMCs. (D) Northern blot analysis indicated a decrease in miR-132 level in whole brain tissue. Each QPCR analysis was performed in triplicate, and expression levels were normalized to U6 snRNA. Error bars represent standard deviations. Northern blot analysis was performed for miRNA detection using the total RNA pool isolated from PBMCs or whole brain tissue of SCZ-like rats (n = 15) and normal rats (n = 15). ∗P b 0.05.
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Keywords: Biomarker microRNA miR-132 Peripheral blood Schizophrenia
School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, PR China Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine in Henan Province, Xinxiang 453003, PR China The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang 453002, PR China d School of Pharmacy, Xinxiang Medical University, Xinxiang 453003, PR China e Department of Psychology, Xinxiang Medical University, Xinxiang 453003, PR China f Department of Pharmacology, School of Medicine, Zhengzhou University, Zhengzhou 450001, PR China
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Hai-chuan Yu a,b,c,f, Jiao Wu d, Hong-xing Zhang e,c, Gao-li Zhang a, Juan Sui a, Wen-wen Tong a, Xin-Ya Zhang a, Li-li Nie c, Ju-hong Duan a, Li-rong Zhang f, Lu-xian Lv c,⁎ c
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Alterations in microRNAs (miRNAs) have been considered to have diagnostic implications in most diseases, but few studies have reported dysregulated miRNAs in schizophrenia (SCZ). In order to observe an association between miRNAs and SCZ, this study was designed to investigate expression profiling of miRNAs in peripheral blood mononuclear cells (PBMCs). miRNA microarray technology was employed to compare the expression of miRNAs in PBMCs from SCZ patients (n = 105) and normal controls (n = 130), and real-time quantitative polymerase chain reaction (QPCR) was used to analyze the results. Several important miRNA levels were examined before and after antipsychotic treatment in first-onset SCZ patients. In addition, an SCZ-like rat model was established using dizocilpine (MK-801), and miR-132 expression in PBMCs and whole-brain tissue from SCZlike rats was studied using QPCR. In humans, dysregulated miRNAs were observed before treatment and QPCR verified that miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 levels were significantly decreased (P b 0.01 for all) in PBMCs of SCZ patients compared with healthy controls. After antipsychotic treatment there was a marked increase in miR-132 (P b 0.01), miR-664⁎ (P b 0.05) and miR-1271 (P b 0.05) levels in SCZ patients compared with the levels before treatment. In the animal assays, miR-132 levels declined in PBMCs and wholebrain tissues (both P b 0.05) of the SCZ-like rats compared to controls. For the first time, our results suggest that miR-132 is a potential and superior biomarker in peripheral blood that will allow discrimination of SCZ patients from healthy controls. © 2015 Published by Elsevier Inc.
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development, failure to develop appropriate neuronal phenotype, neuronal atrophy, severe growth defects and early death (Schaefer et al., 2010). miR-133b regulates the maturation and function of midbrain dopamine neurons through a negative feedback circuit involving the transcription factor pituitary homeobox 3 (Pitx3) (Tognini et al., 2011). miR-124, which promotes neuronal gene expression in differentiating neural progenitor cells, is enriched in mammalian brain. There is a feedback regulatory loop between miR-9, a brain-specific miRNA, and the nuclear receptor TLX, which plays an important role in maintaining neural stem cells in the proliferative and self-renewable state during neural stem cell differentiation (Zhao et al., 2009). miR-132 and miR-134 may functionally control synaptic plasticity, and transcription factor myocyte enhancing factor 2 (Mef2) increases miR-134 expression in response to neuronal activity (Fiore et al., 2009). All of these studies have revealed dysfunction in miRNA signaling contributes to neurodegenerative and psychiatric disorders. Schizophrenia (SCZ) is a severe psychiatric disorder that has an average life time development risk of 0.7%; heritability of the disease is
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1. Introduction
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MicroRNAs (miRNAs) are 20–25-nucleotide, single-stranded, noncoding RNA molecules that play a pivotal role in diverse cellular processes including cell-cycle control, immunity, viral or bacterial disease, stem-cell differentiation, development, metabolism and oncogenesis (Cech and Steitz, 2014; Lujambio and Lowe, 2012; Wu et al., 2014). Accumulating evidence supports a key role for miRNAs in neurogenesis, neuronal maturation and brain development (Belzeaux et al., 2012; Im and Kenny, 2012; Kapsimali et al., 2007; Sun and Shi, 2014a). In zebrafish, deletion of Dicer results in defects in neural development, and injection of miR-430 can rescue many of these defects (Giraldez et al., 2005). Dicer-deficient mice display marked deficits in brain
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⁎ Corresponding author at: Department of Psychiatry of the Second Affiliated Hospital of Xinxiang Medical University, No.388, Jianshe Middle Road, Xinxiang 453002, PR China. Tel.: +86 373 3373969. E-mail address: [email protected] (L. Lv).
http://dx.doi.org/10.1016/j.pnpbp.2015.05.007 0278-5846/© 2015 Published by Elsevier Inc.
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xinxiang Medical University, and written informed consent was obtained from all participants. Between August 2011 and June 2014, 105 patients with SCZ (50 males and 55 females; mean age: 25.03 ± 8.34 years) and 130 healthy controls (60 males and 70 females; mean age: 22.73 ± 6.79 years) were recruited. Individuals with a history of severe medical complications, organic brain disease, any concomitant major psychiatric disorders, or substance dependence were excluded. The consensus diagnoses for SCZ patients were made by at least two psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition IV (DSM-IV). The clinical features of 105 patients were evaluated by the senior psychiatrist using Positive and Negative Symptom Scale (PANSS). During the same period the first-onset schizophrenia patients, who were Chinese of Han descent, were selected and treated with risperidone initially. According to the principle of treatment in our study, patients who experienced an increase in body weight of more than 10%, serious side effects, or no significant improvement in 8 weeks were
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The peripheral blood samples (5 mL per participant) were collected in EDTA anticoagulant tubes and mononuclear cells were separated by density gradient centrifugation using a percoll density gradient (d = 1.077; Amersham Pharmacia Biotech, Germany). Total RNA was extracted from PBMCs with Trizol reagent (Invitrogen, CA, USA) following the manufacturer's instructions and stored at −80 °C until use. For animal assays, blood samples and whole brain tissues were obtained and RNA was isolated in the same manner as above. Total RNA was extracted from different PBMC samples using TRIzol reagent (Invitrogen) and quantified respectively. DNA was removed by treatment with 5 units of DNase I (Promega, Madison, WI) at 37 °C for 45 min followed by inactivation at 65 °C for 10 min. Then firststrand of cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instruction. Stemloop reverse transcription and QPCR for miRNAs were executed as described previously (Chen et al., 2005). cDNA was synthesized from 2 μg of total RNA using reverse transcribed primers as shown in Supplementary Table 1.
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excluded. In addition, some patients were lost to follow-up because of low compliance or noncooperation. After screening, only 10 patients maintained and fulfilled the monotherapy regimen and achieved remission. PANSS was used to evaluate the severity of SCZ and clinical efficacy of treatment. Risperidone was given once daily, with an initial dose of 2 mg/day, increasing up to 3–6 mg/day over the following weeks. The maintenance dose was adjusted according to side effects and clinical assessment. All patients received oral risperidone treatment after baseline assessments and achieved remission after 8 weeks of treatment.
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estimated at 80% based on twin studies (Sullivan et al., 2003; Wright et al., 2013). Although the pathogenetic mechanisms leading to SCZ are unclear, it is well accepted that SCZ is the result of a complex interplay between genetic variants and environmental factors. Recently, there has been much interest in the possible involvement of miRNAs in SCZ (Im and Kenny, 2012; Sun and Shi, 2014a). The findings of the largest Genome-Wide Association Study (GWAS) on SCZ have revealed that the strongest association of SNP with SCZ lies within the intron of a putative primary transcript for miR-137 (Hill et al., 2014; Schizophrenia Psychiatric Genome-Wide Association Study, 2011). Xu et al. (2010) have indicated that miR-30e ss178077483 plays a role in SCZ susceptibility. In animal studies, Kocerha et al. (2009) have found that miR219 mediates the behavioral effects of dizocilpine (MK-801) treatment in mice. An in situ hybridization study has revealed miR-185 expression in brain regions is implicated in SCZ mice, and gene-based tests revealed that converse expression patterns for miR-185 and its target genes (ATAT1, SH3PXD2A, NTRK3) provided no further evidence of their involvement in SCZ. miR-181b, miR-30e, miR-34a and miR-7 are probably involved in the pathogenesis of SCZ, and significant downregulation of miR-181b expression has been observed after treatment with atypical antipsychotics as an antipsychotic response (Song et al., 2014). Miller et al. (2012) have suggested that miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes, DNMT3A, GATA2 and DPYSL3, contribute to the neurodevelopmental and neuromorphological pathologies present in SCZ. Despite growing evidence showing a relationship between altered expression of miRNAs and SCZ, it is unclear which miRNA has the greatest influence upon SCZ and which miRNA can be considered a diagnostic biomarker. In this study, we tried to identify miRNAs that are clinically practicable biomarkers for SCZ through blood-based detection. We compared the miRNA expression profiles of peripheral blood mononuclear cells (PBMCs) by using miRNA microarray technology and real-time quantitative polymerase chain reaction (QPCR), and several markedly different expression levels of miRNAs were observed. Of importance, downregulation of miR-132 in PBMCs stands out in accordance with Miller's results (Miller and Zeier, 2012), which showed that miR-132 was significantly decreased in the prefrontal cortex of SCZ patients. In addition, miR-132 was involved in the pathogenesis of SCZ and significant upregulation of miR-132 expression was found in SCZ patients' PBMCs after antipsychotic treatment. Animal studies demonstrated miR-132 downregulation is associated with SCZ-like animals after MK-801 treatment. All of these results proved that miR-132 may be a potential biomarker in peripheral blood for the diagnosis of SCZ.
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Sixty total RNA samples from PBMCs of SCZ patients and 60 total RNA samples from normal controls were selected and pooled to 6 case pools and 6 control pools (every 10 samples RNA were mixed equivalently in one tube as one pool), respectively. Global miRNA expression profiling of SCZ patients and normal controls was investigated using miRNA microarray (Agilent Human miRNA V19.0) that contained capture probes for 2006 human miRNAs that were registered and annotated in the Sanger miRBase V19.0.
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QPCR was carried out using the SYBR Premix Ex Taq kit (Takara, Dalian, China) according to the manufacturer's instructions using QPCR primers (Supplementary Table 1). U6 snRNA was used as the endogenous control and the comparative Ct method was used to quantify target genes relative to their endogenous control. Each reaction was performed in triplicate. For each individual analysis, one of the samples (from one healthy subject) was designated as the calibrator and given a relative value of 1.0. All quantities were expressed as n-fold relative to the calibrator.
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Northern blot analysis of miRNAs was performed as described previously with minor modifications (Wang et al., 2011). Total RNA was subjected to 15% TBE–Urea gels and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Germany) and crosslinked by ultraviolet irradiation. A γ-32P end-labeled miRNA probe of miR-132 was employed, and U6 snRNA was used as a control probe to verify equal RNA loading.
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Quantified microarray signal intensity values were normalized to per-chip mean values. Differentially expressed miRNAs were identified using the two-class paired t test procedure within significant analysis of microarrays (SAM) with a 5% false discovery rate (FDR) threshold. Cluster and Java TreeView were used to build the unsupervised tree. The genes and arrays were mean-centered, and hierarchical trees were built using correlation metrics. Statistical analyses of QPCR data were performed with the GraphPad Prism 6 and SPSS version 21.0 (SPSS, Chicago, IL). For each pair of sample types, a two-sample t test assuming equal variance was carried out for miRNAs. We used a conventional receiver-operating characteristic (ROC) curve to analyze miRNA levels and to determine the cutoff points that yielded sensitivity and specificity. We calculated the area under the curve (AUC) and 95% confidence intervals for the AUC. The association with statistical significance was assessed by nonparametric tests.
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In our study, all patients in the study group suffered from SCZ. Their age, gender, smoking history, ethnicity and educational level were recorded, and the clinical characteristics and demographics of the SCZ and control groups are shown in Supplementary Table 2. No significant differences were found between the SCZ patients and healthy controls in age (t = 0.023, P = 0.87) or sex (χ2 = 0.104, P = 0.84). At the same time, to investigate the changes in expression of miRNAs before and after antipsychotic therapy, the first-onset SCZ patients (n = 12) were selected and treated with risperidone initially.
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To investigate the potential involvement of miRNAs in SCZ, we scanned global miRNA expression profiles of PBMCs from 60 SCZ patients (6 pools) and 60 healthy controls (6 pools) using miRNA microarray for mature human miRNAs. Following comparison between the SCZ patients and healthy controls, we identified certain miRNAs, the majority of which showed decreased expression in SCZ patients. Among them, we focused on 41 downregulated miRNAs (Fig. 1), whose expressions were significantly decreased in SCZ patients, and the change folds were all greater than 2.5 compared with controls (Supplementary Table 3).
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To verify the accuracy and universality of the miRNA microarray, QPCR analysis was performed on the differentially expressed miRNAs of PBMCs in expanded samples, including 105 SCZ patients and 130
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Male Sprague-Dawley rats (200–250 g) were provided by the Experimental Animals Center of Xinxiang Medical University. All experimental protocols were reviewed and approved by the Animal Care and Use Committee of Xinxiang Medical University. Thirty SD rats were randomly assigned to two batches: a chronic MK-801-induced SCZ-like rat model (n = 15) was established by repeated intraperitoneal administration of MK-801 (dissolved in saline, 0.3 mg/kg/day), and rats that received saline once a day for 4 weeks served as controls (n = 15). After 4-week administration of MK-801, an open field test (total distance traveled, cm) and social interaction assay were performed as described previously (Guo et al., 2010; Huang et al., 2014). Rats were injected with MK-801 or saline 30 min prior to behavioral tests and were put into an open arena and measured.
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Fig. 1. Differential expression of miRNAs in SCZ patients and healthy controls. Unsupervised hierarchical clustering analysis of miRNA expression profiles in 6 pools from PBMCs of SCZ patients and 6 pools from healthy controls. The degree of relatedness of the expression patterns in the samples is represented by the dendrogram at the left of the panel. Each column corresponds to the expression profile of a sample, and each row corresponds to a miRNA. The color in each cell reflects the level of expression of the corresponding miRNA in the corresponding sample. Increasing intensities of red mean that a specific miRNA has a higher expression in the given sample and increasing intensities of green mean that this miRNA has a lower expression. The scale reflects the miRNA abundance ratio in a given sample relative to the mean level for all samples. The red arrow presents the focus miRNAs of this paper. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
healthy controls. Average expression levels of individual tested miRNAs from SCZ patients and healthy controls are shown in Supplementary Fig. 1; expression levels of all selected miRNAs of SCZ patients were compared with those of healthy controls. From these QPCR results, a significant decrease (P b 0.01) in miRNA level was noted in PBMCs of SCZ patients for six miRNAs (miR-132, miR-664⁎, miR-1271, miR-200c, miR-432, miR-134). One of the normal healthy samples (N-1) was designated as the calibrator and the relative expressions of other samples were calculated by comparing the individual expressions of all miRNAs with those of this calibrator. We measured the expression levels of the miRNAs for the SCZ patients and healthy controls, and found that the relative expression levels of miR-132 (median: SCZ 0.59, healthy controls 1.30, P b 0.01, Fig. 2A), miR-134 (median: SCZ 0.54, healthy controls 1.37, P b 0.01, Fig. 2B), miR-1271 (median: SCZ 0.67, healthy controls 1.66, P b 0.01, Fig. 2C), miR-664⁎ (median: SCZ 1.10, healthy controls 1.70, P b 0.01, Fig. 2G), miR-132 (median: SCZ 0.64, healthy controls 1.46, P b 0.01, Fig. 2H) and miR-432 (median: SCZ 0.62, healthy controls 1.2, P b 0.01, Fig. 2I) were significantly lower in SCZ patients than those in healthy controls. No significant differences in other miRNA levels were observed between SCZ patients and healthy control
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Fig. 2. Discrimination of miRNA levels in PBMCs of SCZ patients (n = 105) and healthy controls (n = 130). (A), (B), (C), (G), (H), and (I): miR-132, miR-134, miR-1271, miR-664⁎, miR200c, and miR-432 expressions, respectively, in PBMCs of SCZ patients and healthy controls. There were significant decreases in miR-132, miR-134, miR-1271, miR-664⁎, miR-200c, and miR-432 in PBMCs of SCZ patients (P b 0.01). (D), (E), (F), (J), (K), and (L): ROC curves were drawn for miR-132, miR-134, miR-1271, miR-664⁎, miR-200c, and miR-432, which yielded 0.934, 0.920, 0.921, 0.836, 0.878, and 0.821 as the AUC values, respectively.
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groups, indicating the uniqueness of miR-132, miR-664⁎, miR-1271, miR-200c, miR-432 and miR-134 as SCZ biomarkers. ROC analysis was subsequently used to assess the diagnostic sensitivity and the specificity (Fig. 2). Diagnostic sensitivities of miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 for SCZ were 94%, 92%, 91%, 88%, 93% and 88%, respectively. The corresponding specificities were 89%, 82%, 81%, 74%, 78% and 68%, and ROC curve areas (95% CI) were 0.934 (89.4–97.5%), 0.920 (88.0–96.0%), 0.921 (88.1–96.1%), 0.836 (78.0–89.1%), 0.878 (82.8–92.7%) and 0.821 (76.5–87.6%), respectively (Table 1). The data showed the significant diagnostic value of
miR-132, miR-134, miR-1271, miR-664⁎, miR-200c and miR-432 for 278 SCZ, and these miRNAs may be novel noninvasive biomarkers for the 279 diagnosis of SCZ. 280 3.4. miRNA expression differences before and after antipsychotic treatment 281 in first-onset SCZ patients 282 To investigate the changes in these miRNA levels before and after an- 283 tipsychotic treatment, blood samples were collected on the day before 284 (baseline) and the day after 8 weeks of risperidone treatment (end 285
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Specificity (%)
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0.945 0.958 1.169 1.402 1.050 0.911
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Receiver-operating characteristic (ROC) curve analysis was used to determine the cutoff points that yielded the highest combined sensitivity and specificity. The relative expression of miRNA copies to U6 snRNA copies were used to perform the ROC curve analysis.
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point) and used for QPCR detection. Details are provided in Supplementary Table 4. Risperidone is one of the most widely used first-line antipsychotics for acute SCZ. We measured the expression levels of these six miRNAs in peripheral blood of SCZ patients before and after risperidone treatment, and the results are shown in Fig. 3. According to these data, the expression of miR-132 was significantly upregulated after risperidone treatment compared to before risperidone treatment (P b 0.01) (as in Fig. 3A), while the levels of miR-1271 and miR-664⁎ were also moderately higher (P b 0.05) after risperidone treatment (as in Fig. 3C and D), but there were no significant differences in the levels of miR-134, miR-200c and miR-432. These results indicated that risperidone improved certain miRNAs levels in SCZ patients significantly; in particular, miR-132 may potentially be involved in a therapeutic strategy as an effective biomarker.
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3.5. Changes in expression of miR-132 in the MK-801 SCZ-like rat model
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In animal assays, locomotor activity was recorded for 30 min as an indicator of “SCZ-like” behavior. MK-801 treated rats (n = 15) exhibited significantly increased locomotor activity compared with salinetreated controls (n = 15), as expected. At the same time, social interaction was tested through videotaping for 20 min and a decrease in social interaction duration was observed in the MK-801 group compared with the saline group (Supplement Fig. 2). Therefore, the MK-801 SCZ-like rat model was established successfully. QPCR and Northern blot results
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Multiple studies have shown that defective neuronal plasticity and function in neurodevelopmental disorders may have resulted from impaired posttranscriptional regulation mediated by miRNAs (Hunsberger et al., 2009) and that miRNAs play important roles in the etiology of neuropsychiatric and neurodevelopmental disorders, and neurodegenerative diseases (Olde Loohuis et al., 2012; Sun and Shi, 2014b). miR132 expression has been reported to be dysregulated in Alzheimer's disease and SCZ (Cogswell et al., 2008). Kim et al. (2010) have proved that miR-132 expression is correlated with SCZ by examining the postmortem prefrontal cortex of patients with SCZ. Miller and Zeier, (2012) have examined the expression of miRNAs in prefrontal cortical tissue from 100 control and schizophrenic subjects and found that miR-132 was significantly downregulated in SCZ patients compared with controls. Now, we have observed a significant decrease in miR132 in peripheral blood of SCZ patients, and these data are in accordance with Miller's results in prefrontal cortical tissue. The reduced levels of miR-132 in peripheral blood and prefrontal cortical tissue may be reflecting the same characteristic of SCZ, and all of these results illustrated that miR-132 is a potential target for diagnosis and treatment of diseases related to SCZ. Recent studies have suggested that aberrant expression of miRNAs is a potential diagnostic biomarker of disease (Jansen et al., 2014; Li et al., 2013; Li et al., 2014; Silva et al., 2014). Since brain tissue is not readily accessible, a new focus in research on biomarkers is blood-based expression profiling of miRNAs. For blood-based miRNA detection, PBMCs are more stable and feasible to use than serum because the contents of miRNAs in serum are very low. However, it has been unclear which miRNAs in human peripheral blood can be regarded as biomarkers for SCZ, and how the expressions of these miRNAs change before and after antipsychotic therapy. In this study, miRNA microarray was employed to identify the differences in miRNA expression in peripheral blood between SCZ patients and healthy controls, and 41
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4. Discussion
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Cut-off point
0.934 0.920 0.921 0.836 0.878 0.821
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0.894–0.975 0.880–0.960 0.881–0.961 0.780–0.891 0.828–0.927 0.765–0.876
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miR-132 miR-134 miR-1271 miR-664⁎ miR-200c miR-432
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indicated that the expression level of miR-132 was significantly lower in 309 PBMCs (Fig. 4A and C) and whole brain tissue (Fig. 4B and D) of the SCZ- 310 like rats than in the normal control group. 311
Table 1 Diagnostic accuracy based on PBMC miRNA levels.
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Fig. 3. (A–F) Comparison of miRNA levels in PBMCs before and after 8 weeks of risperidone treatment in first-onset SCZ patients (n = 10) using QPCR. Significant increases in miR-132 (P b 0.01), miR-1271 (P b 0.05), and miR-664⁎ (P b 0.05) were found after treatment when compared with before.
Please cite this article as: Yu H, et al, Alterations of miR-132 are novel diagnostic biomarkers in peripheral blood of schizophrenia patients, Prog Neuro-Psychopharmacol Biol Psychiatry (2015), http://dx.doi.org/10.1016/j.pnpbp.2015.05.007
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Contributors
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Hai-chuan Yu, Lu-xian Lv, and Li-rong Zhang designed the study. Hai-chuan Yu and Jiao Wu wrote the first draft of the manuscript. Haichuan Yu, Wen-wen Tong, Gao-li Zhang, Gao-li Zhang, and Juan Sui performed the experimental work. Jiao Wu and Hong-xing Zhang undertook statistical analyses. Li-li Nie and Ju-hong Duan undertook all sample collections and support. All authors contributed to and have approved the final manuscript.
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(No. 13B350215, No. 14B320015), Foundation for Excellent Innovation Talents of Health Science and Technology by Henan Health Department (No. cxyx20130418) and Scientific Research Fund Project of Xinxiang Medical University (No. 100403, No. 505002).
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Role of funding source
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This work was financially supported by the National Natural Sciences Foundation of China (No. 31301135), Science and Technology Research Projects of the Education Department of Henan Province
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The authors wish to thank all the subjects who participated in the study. Authors also acknowledge invaluable assistance by numerous mental health professionals in the different clinical departments of the Second Affiliated Hospital of Xinxiang Medical University.
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References
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downregulated miRNAs were found. Then, QPCR was used to validate the data from miRNA microarray, and six miRNAs (miR-132, miR664⁎, miR-1271, miR-200c, miR-432 and miR-134) exhibited significant decreases (P b 0.01) in peripheral blood of 105 SCZ patients compared to 130 healthy controls. To investigate whether the six miRNAs may be informative as SCZ therapeutic markers, we compared miRNA expression in peripheral blood from first-onset SCZ patients before and after antipsychotic therapy. We found that the expressions of miR-132, miR-1271 and miR664⁎, but not miR-134, miR-200c and miR-432, increased in PBMCs after risperidone treatment compared to before treatment. In addition, the MK-801 SCZ-like model was established successfully and the six miRNAs were detected. QPCR and Northern blot analysis demonstrated that miR-132, but not the other miRNAs, was reduced in PBMCs or whole brain tissue of rats with MK-801 treatment compared to normal saline controls. Based on these findings, we suggest that miR-132 could be a superior marker for detection of SCZ by blood tests. Indeed, the miR-132 level decreased going from non-SCZ patients to SCZ patients and was markedly increased after risperidone treatment. In further study, the important target genes of miR-132 involved in SCZ will be investigated by using dual luciferase reporter assays and Western blot, and the expression changes of these genes in PBMCs from SCZ patients will be measured. In general terms, the identification of SCZ-specific miRNA profiles in PBMCs is an emerging field of particular interest, but it can be stated that the study of miRNAs in SCZ is in its early stages. Only a handful of very recent studies have shed a little light on this growing field. Although the role of miRNAs in the clinical management of psychiatric disease has yet to be studied, our work shows that they are exciting potential candidates for this challenge. Taken together, our observations have demonstrated that miR-132 plays an important role as a diagnostic biomarker in SCZ. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pnpbp.2015.05.007.
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Fig. 4. Compared with the normal group (n = 15), the expression level of miR-132 was significantly downregulated in PBMCs and whole brain tissue of SCZ-like rats (n = 15). (A) QPCR analysis of miR-132 expression in PBMCs. (B) QPCR analysis of miR-132 expression in whole brain tissue. (C) Northern blot analysis indicated a decrease in miR-132 level in PBMCs. (D) Northern blot analysis indicated a decrease in miR-132 level in whole brain tissue. Each QPCR analysis was performed in triplicate, and expression levels were normalized to U6 snRNA. Error bars represent standard deviations. Northern blot analysis was performed for miRNA detection using the total RNA pool isolated from PBMCs or whole brain tissue of SCZ-like rats (n = 15) and normal rats (n = 15). ∗P b 0.05.
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