Alterations of microRNA-124 expression in peripheral blood mononuclear cells in pre- and post-treatment patients with major depressive disorder

Journal of Psychiatric Research 78 (2016) 65e71

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Alterations of microRNA-124 expression in peripheral blood mononuclear cells in pre- and post-treatment patients with major depressive disorder Shen He a, Xiaohua Liu a, **, Kaida Jiang a, Daihui Peng a, Wu Hong a, Yiru Fang a, Yiping Qian b, Shunying Yu b, Huafang Li a, b, c, * a b c

Department of Psychiatry, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China Institution of Drug Clinical Trials, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 September 2015 Received in revised form 6 March 2016 Accepted 30 March 2016

Recently, increasing evidence has indicated that dysfunction of microRNA-124 (miR-124) might be involved in the pathophysiology and treatment of major depressive disorder (MDD) in some animal models of depression. However, the role of miR-124 in MDD patients remains unclear. The objective of this study was to investigate whether the miR-124 expression levels in peripheral blood mononuclear cells (PBMCs) were associated with MDD and to evaluate the effects of antidepressant treatment on miR124 levels. Quantitative real-time PCR was applied to detect miR-124 expression in 32 pre- and posttreatment MDD patients and 30 healthy controls. Our results showed that expression levels of miR124 from PBMCs in MDD patients were significantly higher than those in healthy controls (p < 0.001), and that the area under the curve of miR-124 from ROC analysis was 0.762 with a sensitivity of 83.33% and specificity of 66.67% in distinguishing MDD patients from healthy controls. In addition, the expression levels of miR-124 were significantly down-regulated after eight weeks of treatment (p < 0.001). MiRNA target gene prediction and functional annotation analysis indicated that altered miR124 was involved in affecting some important biological processes and pathways related to MDD. These results provide new information on miR-124 involvement in the biological alterations of MDD and in antidepressant effects. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Major depressive disorder MicroRNA miR-124 PBMC Expression Treatment

1. Introduction Despite a few decades of unremitting efforts, the pathogenesis of major depressive disorder (MDD) and the mechanisms of antidepressant treatment have not been elucidated. In recent years, the neurotrophic hypothesis of depression has increasingly attracted the attention of researchers. Briefly, this hypothesis states that the impairment of synaptic plasticity, particularly in the hippocampus, may be a core factor in the pathophysiology of depression (Duman

* Corresponding author. Institution of Drug Clinical Trials, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 600 South Wan Ping Road, Shanghai 200030, PR China. ** Corresponding author. Department of Psychiatry, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 600 South Wan Ping Road, Shanghai 200030, PR China. E-mail addresses: [email protected] (X. Liu), [email protected] (H. Li). http://dx.doi.org/10.1016/j.jpsychires.2016.03.015 0022-3956/© 2016 Elsevier Ltd. All rights reserved.

and Li, 2012). MiRNAs are 22e25 nucleotide non-coding RNAs that can negatively regulate gene expression by repressing mRNA translation and/or mediating cleavage of mRNAs at the posttranscriptional level (Flynt and Lai, 2008). MiRNAs are abundant in the brain, and they have been found to play a critical role in several aspects of brain function, particularly neurogenesis, neuronal plasticity, and neuronal development (Kosik, 2006 and Zeng, 2009). Emerging evidence has demonstrated that dysregulation of miRNA expression occurs in animal models of depression (Cao et al., 2013), in the post-mortem brain tissue of depressed subjects (Lopez et al., 2014 and Smalheiser et al., 2012, 2014), and in the CSF and the peripheral blood of depressed patients (Belzeaux et al., 2012; Fan et al., 2014; Li et al., 2013 and Wan et al., 2015). Moreover, it has been shown that selective serotonin reuptake inhibitors, other types of antidepressants, and other physical treatment such as electroconvulsive therapy, can modify aberrant

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miRNA expression and their downstream targets (Belzeaux et al., 2012; Bocchio-Chiavetto et al., 2013; Lopez et al., 2014 and Ryan et al., 2013). Among miRNAs, miR-124 is the most abundant miRNA in the brain (Lagos-Quintana et al., 2002). MiR-124 is derived from three independent genes (miR-124-1 also known as miR-124a, miR-124-2, and miR-124-3) that all produce the same mature miRNA. It has been reported that miR-124 can regulate adult neurogenesis, promote neuronal differentiation, and contribute to synaptic plasticity in vivo (Cheng et al., 2009 and Makeyev et al., 2007). In a previous study, miR-124 expression levels were found to be increased in the hippocampus of chronic unpredictable stress induced depression model rats (Cao et al., 2013). Similarly, another recent study has shown that overexpression of miR124a contributes to chronic social stress-induced depression in rats. Moreover, this study also found that knockdown of miR124a expression by lentivirus mediated siRNA expression vector (LV-siR124a) in the hippocampus of rats had an antidepressant-like effect (Bahi et al., 2014). In this situation, we could speculate that miR-124 is associated with the pathophysiology of depression and is a potential target for antidepressant treatments. To date, though there have been some studies on the alteration of miR-124 levels in animal models of depression, few clinical studies have investigated the relationship between the levels of miR-124 in PBMCs and MDD patients. In addition, there are few data available yet pertaining to the effect of antidepressants on miR-124 levels in PBMCs in MDD patients. Therefore, in this study, we combined a cross-sectional and longitudinal naturalistic observational design to determine the possible alterations of miR124 levels of PBMCs in MDD patients prior to treatment by comparing those in healthy controls and to further investigate whether antidepressant treatment can change the miR-124 levels in MDD patients. On these bases, we sought to explore the potential role of miR124 in biological alterations of MDD and in antidepressant effects. Putative target genes of the miR-124 and their possible functions and biological mechanisms were investigated using bioinformatics tools. 2. Materials and methods 2.1. Participants Thirty two in- and out-patients with MDD were included in this study from the Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine. All diagnoses were made according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV) using the Structured Clinical Interview for DSM-IV (SCID). The inclusion criteria were as follows: (1) age 18e60 years, (2) Han ethnicity, (3) either medication-naive or medication-free for at least eight weeks before recruitment, and (4) Hamilton Depression Rating Scale (HDRS-24)
20. Patients with any Axis-I psychiatric disorders other than MDD were excluded from this study. Thirty age- and sex-matched healthy volunteers of Han ethnicity were recruited as controls. Healthy controls (HC) were evaluated using the Structured Clinical Interview for DSM-IV disordersdNon-patient Version (SCID-I/NP). No first-degree relatives of healthy control subjects had any Axis-I psychiatric disorder. All participants received a clinical interview, laboratory tests and a physical examination to rule out physical illnesses. In addition, the medical records of all participants were reviewed. Any participants with any physical diseases (i.e., organic brain diseases, hypertension, diabetes mellitus, tumor, severe infectious or inflammatory diseases and thyroid disease) were excluded from this

study. Participants over the age of 60 years were excluded because there was a greater chance of other co-morbid physical illness as well as psychological issues associated with old age. In addition, pregnant or lactating participants were excluded. The Institutional Review Board of Shanghai Mental Health Center approved the research protocol, and all participants provided written informed consent. 2.2. Naturalistic follow-up and clinical assessment All of the patients received individually tailored pharmacotherapy according to the currently accepted therapeutic guidelines from the American Psychiatric Association (American Psychiatric Association, 2010). The HDRS-24 was used to evaluate the severity of symptoms in MDD patients at baseline (w0) and after eight weeks of treatment (w8). The efficacy of antidepressant treatment was evaluated according to the reduction rate of the total HDRS score. Response to treatment was considered a reduction in HDRS score of at least 50%. 2.3. Blood collection and RNA extraction Whole blood (5 ml) was collected from each subject using EDTA anticoagulant tubes and processed within 2 h. PBMCs were isolated from whole blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. Then, PBMC material was transferred into fresh RNase/DNase-free 2 ml microcentrifuge tubes and stored at 80  C until further use. Total RNAs were extracted from the PBMCs using mirVana TM miRNA Isolation (Applied Biosystems, p/nAM1560) according to the manufacturer's protocol. RNA integrity was checked by agarose gel electrophoresis and the ratio of optical density (OD) at 260 nm and 280 nm was also measured. RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific). 2.4. MiRNA analysis Levels of miR-124 (TaqMan Assay ID: 001182) were measured individually in each sample using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. A TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to perform reverse transcription (RT) of the total RNA samples. Each reverse transcription reaction consisted of 5 mL of total RNA (10 ng), 1.5 mL 10X RT buffer, 0.15 mL of 100 mM dNTPs (with dTTP), 0.19 mL of RNase inhibitor (20 U/mL), 1 mL of MultiScribe Reverse Transcriptase (50 U/mL), 4.16 mL nuclease-free water and 3 mL 5  RT primer (final volume 15 mL). The mixture was incubated at 16  C for 30 min, 42  C for 30 min and 85  C for 5 min and then held at 4  C. Following the RT reaction, real-time PCR was conducted using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Inc., USA) with 20 mL PCR reaction mixture that contained 1.33 mL of RT product, 1 mL of 20  TaqMan Individual microRNA assay, 10 mL of 2  TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA, USA) and 7.67mL of nuclease free water. PCR reactions were run at 50  C for 2 min and at 95  C for 10 min, followed by 40 cycles at 95  C for 15 s and at 60  C for 1min in 384well plates. All reactions were performed in triplicate, including negative controls. Raw Ct values were collected using the SDS 2.3 software (Applied Biosystems, Inc.). U6 snRNA (Assay ID: 001973) was used as an endogenous control to normalize miR-124 expression in PBMCs. A previous study showed U6 sn RNA has relatively high expression and the smallest variation (standard deviation of mean Ct < 0.5) compared to other miRNAs and non-coding RNAs expressed in PBMCs in both MDD patients and healthy controls (Belzeaux et al., 2012). This was also

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confirmed by our data. The standard deviation of mean U6 Ct was below 0.5 in this study. In addition, there was no significant difference in U6 Ct values between MDD patients at baseline and healthy controls (ManneWhitney U test: Z ¼ 1.183, p ¼ 0.237). Moreover, no significant difference was observed in U6 Ct values between MDD patients at baseline and MDD patients after eight weeks of treatment (paired t-test: t ¼ 0.499, p ¼ 0.622). These results suggested that the expression of U6 was stable in our PBMC samples. The Ct values for miR-124 were 33.48 ± 1.025 for all samples, and all samples had a Ct value below 36. All negative controls included no template control, and no RT control had undetectable Ct and no amplification. After normalization by U6 snRNA, the relative expression levels of miR-124 were calculated using the 2DDCt method (Schmittgen and Livak, 2008). For each individual analysis, the particular sample (either the sample of the highest DCt (Ct miR-124- Ct U6) from control subjects when comparing patients to controls, or the week 0 sample of the highest DCt from MDD patients when comparing patients at week 0 to patients at week 8) was designated as the calibrator and given a relative value of 1.0. All other quantities were expressed as the n-fold difference relative to the calibrator. 2.5. Target prediction and pathway analysis We obtained the predicted target genes of miR-124 from miRecords, which stores predicted miRNA targets produced by 11 different established miRNA target prediction algorithms, including DIANA-microT, MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22, RNAhybrid, and TargetScan (Xiao et al., 2009). To ensure accurate prediction, only those targets predicted by at least four different algorithms in miRecords were included in the present study. Moreover, to identify miR-124 targets genes associated with psychiatric disorders, we compared the two datasets from miRecords and DisGeNET database. DisGeNET is a new gene-disease database integrating information on genedisease associations from several public data sources and the ~ ero et al., 2015). In this study, the “mental disorders” literature (Pin (umls: C0004936) was used as query for the DisGeNET database. The genes commonly shared by miRecords and DisGeNET database were identified to be associated with psychiatric disorders. In addition, gene ontology (GO) biological processes and Kyoto encyclopedia of genes and genomes (KEGG) pathways enriched in the predicted miRNA target gene datasets were identified with DAVID (version 6.7) (da Huang et al., 2009) using the criteria that at least ten genes were involved and that p < 0.05 for each category. 2.6. Statistical analysis Categorical variables were assessed using Chi-square tests or Fisher exact test, such as for gender. Continuous variable were first tested for normal distribution by the ShapiroeWilk test. The age and BMI were compared between patients and controls using an independent-samples t-test. Expression levels of miR-124 all followed the asymmetrical distribution in controls (w0) and patients (both w0 and w8). The differences in miR-124 expression levels between MDD patients before treatment and healthy controls were compared using the ManneWhitney U test. In addition, the Wilcoxon test for paired samples was used to evaluate the change of miR-124 expression levels in all MDD patients after treatment. Furthermore, Spearman's correlation coefficients were calculated to assess the relationship between miR-124 expression levels and clinical variables. Receiver operating characteristic (ROC) curve analysis, using the relative expression level of miR-124 as the input data, was applied to assess the discriminatory power of miR-124 in distinguishing MDD patients from healthy controls. All results are

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presented as the mean ± standard deviation (SD). Statistical significance was defined as p < 0.05 (two-tailed). All of the statistical analyses were performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Naturalistic follow-up The demographic and clinical data of the participants are listed in Table 1. After eight weeks, all of the patients completed the follow-up evaluation and sample collection. The following antidepressants were administered: venlafaxine (N ¼ 7), paroxetine (N ¼ 7), fluoxetine (N ¼ 3), escitalopram (N ¼ 11), duloxetine (N ¼ 1), sertraline (N ¼ 3) and mirtazapine (N ¼ 2). All of the patients received antidepressant monotherapy, except for one patient who received an antidepressant plus electroconvulsive therapy (ECT), one patient who received an antidepressant plus benzodiazepines, and two patients who received combined treatment with two types of antidepressants. As shown in Table 2, the depressive symptoms of the patients improved significantly at the end of study, as indicated by a reduction in HDRS-24 total score after eight weeks of antidepressant treatment (w0 ¼ 27.81 ± 5.65; w8 ¼ 7.38 ± 6.46; Z ¼ 4.94, p < 0.001). Of all patients, 26 patients were responders, and the other six patients were non-responders. The median HDRS scores were reduced by 81.25%. 3.2. Higher levels of miRNA-124 from PBMCs in patients with MDD As shown in Fig. 1, the expression levels of miR-124 were significantly higher in the MDD group than those in the control group (10.18 ± 8.19 vs. 4.68 ± 3.16, Z ¼ 3.747, p < 0.001). Additionally, as seen in Fig. 1, among all of the samples from the 32 MDD patients, two of them had showed extremely high relative expression levels of miR-124. In addition, miR-124 levels in these same two samples dramatically dropped at week 8 (Fig. 3). Therefore, these two samples were removed for more precise analysis. Moreover, removal of them did not affect the results reported here when comparing patients to controls (8.57 ± 5.32 vs. 4.68 ± 3.16, Z ¼ 3.489, p < 0.001). After removing these two samples, we found that the miR-124 levels at baseline were not correlated with age in across participants (r ¼ 0.009, p ¼ 0.947), in the MDD group (r ¼ 0.061, p ¼ 0.749) and in the HC group (r ¼ 0.075, p ¼ 0.693). In addition, the baseline miR-124 levels were not significantly correlated with baseline total HDRS scores (r ¼ 0.132, p ¼ 0.488). ROC curve analysis was used to determine Table 1 Demographic and clinical characteristics of the participants.

Sex (M/F) Age (years) BMI Age of onset (years) Length of disease (months) Patients With first episode With recurrent episode Patients With familial history Without familial history

MDD (n ¼ 32)

HC (n ¼ 30)

c2 or t

P Value

12/20 34.41 ± 21.65 ± 30.16 ± 46.00 ±

11/19 35.60 ± 10.00 21.95 ± 2.92

0.005 0.448 0.425

0.946a 0.656b 0.673b

10.90 2.64 11.71 59.33

15 17 8 24

MDD: major depressive disorder; HC: healthy controls. a Chi-square test. b Independent samples t-test.

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Table 2 Means of HDRS scores and miR-124 levels in MDD patients before and after treatment.

w0 (mean ± SD) w8 (mean ± SD) w0 vs. w8

HDRS scores

MiR-124 levels (RQ)

27.81 ± 5.65 7.38 ± 6.46 p < 0.001a,b

4.17 ± 3.36 1.86 ± 1.48 p < 0.001a,b

w0: before treatment; w8: after 8-week treatment. a Wilcoxon signed-rank test. b Significant difference (p < 0.05).

(AUC) of miR-124 was 0.762 (95% confidence interval [CI] 0.641e0.884). In addition, our results showed that miR-124 had a relatively high sensitivity of 83.33% and a low specificity of 66.67%. The low specificity indicated that miR-124 might have no values for diagnosing MDD. 3.3. Changes in expression of miRNA-124 in MDD patients following treatment As shown in Fig. 3, at the end of the eight weeks of treatment, the Wilcoxon test analysis showed that the miR-124 levels in the 32 patients were significantly decreased compared with those at baseline (4.17 ± 3.36 vs.1.86 ± 1.48, Z ¼ 3.571, p < 0.001) (Fig. 3; Table 2). Likewise, for more precise analysis, we removed those two samples mentioned above for further analysis. After removing them, the paired t-test was used to analyze miR-124 levels in the pre- and post-treatment MDD groups because the differences between miR-124 levels at w0 and w8 were normally distributed. We found that the miR-124 levels were still significantly decreased after treatment compared to baseline levels (3.52 ± 3.36 vs.1.86 ± 1.48, t ¼ 3.284, p ¼ 0.003). 3.4. Response analysis

Fig. 1. The miR-124 expression levels in MDD patients and healthy controls at baseline. The bar represents the median, p < 0.001 (Mann-Whitney U test).

the diagnostic value of miR-124 in discriminating MDD patients from healthy controls. As shown in Fig. 2, the area under the curve

The reduction rate of HDRS scores was significantly correlated with the changes in miR-124 levels (DCt w8 - DCt w0) after the eight weeks of treatment using Spearman's correlation (r ¼ 0.365, p ¼ 0.047). Furthermore, we conducted a subgroup analysis. Patients were divided into responders and non-responders according to the reduction rate of the total HDRS score. Among the remaining 30 MDD patients, 24 patients were responders and the other 6 patients were non-responders. We found the levels of miR-124 declined significantly in responder patients (t ¼ 3.112, p ¼ 0.005) but not in non-responder patients (t ¼ 1.090, p ¼ 0.325). In addition, there were no significant differences in age (t ¼ 0.883, p ¼ 0.385), sex (Fisher exact test: p ¼ 0.620), baseline HDRS score (t ¼ 0.2, p ¼ 0.843) or baseline miR-124 levels (t ¼ 0.402, p ¼ 0.690) between responder patients and nonresponder patients. 3.5. Target gene prediction and pathway analysis of miRNA-124 A total of 1329 target genes predicted by at least 4 different of 11 algorithms in miRecords were remained and these target genes were subjected to GO analysis. Several target genes associated with

Fig. 2. Receiver operating characteristic (ROC) analysis of miR-124 for discriminating MDD patients from healthy controls. MiR-124 yields an area under the ROC curve of 0.762 (sensitivity of 83.33% with a specificity of 66.67%).

Fig. 3. MDD patients had a significant decrease in miR-124 expression levels following eight weeks of antidepressant treatment (p < 0.001).

S. He et al. / Journal of Psychiatric Research 78 (2016) 65e71

psychiatric disorders were listed in Table S1. Moreover, some of them such as SLC6A15, MECP2, NR3C2, ATF7IP, CLOCK, HTR2C and FMR1 have been previously reported to be involved in MDD (Iwamoto and Kato, 2003; Klok et al., 2011; Kohli et al., 2011; Kripke et al., 2013; Kishi et al., 2011; Su et al., 2015 and Song et al., 2013). In addition, GO biological process analysis showed these 1329 target genes were significantly enriched in terms “regulation of transcription”, “signal transduction”, “neuron differentiation”, “neuron development”, “regulation of apoptosis”, “axon guidance”, “Wnt receptor signaling pathway”, “regulation of synaptic transmission”, among others (Bonferroni-corrected, p < 0.05) (Table S2). Some of these biological processes have been shown to be involved in the development of depression (Beasley et al., 2002 and Duman, 2002). Likewise, the KEGG analysis showed that a significant enrichment in several pathways related to neuronal brain function of MDD such as axon guidance, long-term depression, long-term potentiation, the ErbB signaling pathway and Insulin signaling pathway (Table S3) (Fan et al., 2014; Gormanns et al., 2011; Kao et al., 2012 and Zubenko et al., 2014). 4. Discussion Because brain tissue is not readily accessible, many studies used peripheral blood samples, mainly including serum/plasma and PBMCs, for basic and clinical research because it is easy to obtain. Several studies have indicated that transcriptional alterations in PBMCs may reflect the molecular and cellular changes in the brain (Fan et al., 2015 and Fisar and Raboch, 2008). The central nervous system (CNS) may exert its influence on the gene expression of peripheral lymphocytes via cytokines, neurotransmitters, or hormones, which may explain the comparable gene expression levels between peripheral blood and some CNS tissues (Gladkevich et al., 2004; Lai et al., 2011 and Sullivan et al., 2006). Moreover, some evidence has demonstrated that PBMCs and brain may share a common miRNA expression pattern (Liang et al., 2007). In addition, recent studies have suggested that aberrant expression of miRNAs in PBMCs samples might be involved in the pathogenesis of MDD (Belzeaux et al., 2012 and Fan et al., 2014). Based on the evidence described above, we chose to use PBMCs for the PCR experiment in the present study. In some previous studies, it has been reported that miR-124 expression levels were increased in the hippocampus of depression models rats, and our findings are in accordance with their results (Bahi et al., 2014 and Cao et al., 2013). However, to our knowledge, this is the first study finding that the alteration of miR124 expression levels from PBMCs in MDD patients before and after antidepressant treatment. In this study, we observed that upregulation of miR-124 expression levels from PBMCs in MDD patients compared to healthy controls. In addition, the miR-124 expression levels in MDD patients decreased significantly after eight weeks of treatment, especially in responders but not in nonresponders. These findings indicate that the alteration of miR-124 expression levels may be associated with MDD and the antidepressant treatment. Our results concerning miR-124 levels are different from the findings in two previous studies. Belzeaux et al. used miRNA Taqman Low Density Array to investigate the alteration of miRNA expression profiles of PBMCs in pre and post-treatment MDD patients (Belzeaux et al., 2012). They did not find the altered levels of miR-124. In contrast, we used the qRT-PCR technology to directly investigate the alterations of miR-124 levels in pre and posttreatment MDD patients. Thus, different quantification method may explain this discrepancy. In addition, in another study, Bocchio-Chiavetto et al. conducted a whole-miRNome quantitative analysis of the changes in the whole blood of 10 depressed subjects

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after 12 weeks of treatment with escitalopram (Bocchio-Chiavetto et al., 2013). They found that 30 miRNAs were differentially expressed after escitalopram treatment. However, no significant differences were found in the miR-124 levels of MDD patients before and after treatment. The discrepancies between their and our results may be accounted for by the difference in quantification method, types of antidepressant drugs and tissue-specific expression pattern (e.g. whole blood/PBMCs). A previous study has demonstrated microRNA levels quantified in whole blood varies from PBMCs (Atarod et al., 2014). To date, though the role of circulating miR-124 in MDD remains unclear, there are a few biological mechanisms that can explain our findings. MiR-124, one of the highly conserved miRNAs, has been shown to regulate growth, proliferation and apoptosis in the CNS. Overexpression of miR-124 results in an increase in the proportion of post-mitotic neurons, a decrease in dividing precursors and a significant decrease in astrocytes (Cheng et al., 2009). Recently, a study indicated that miR-124 can inhibit the expression of BDNF (a validated miR-124 target, Chandrasekar and Dreyer, 2009) in the hippocampus of depression model rats (Bahi et al., 2014), and low expression levels of BDNF in brain tissue are considered to be part of the main pathogenesis of MDD (Ray et al., 2014). Taken together, over-expression of miR-124 may contribute to MDD, at least in part through suppressing BDNF expression. To fully understand the function of the miR-124, we identified the predicted miR-124 targets with miRecords and performed a GO term and KEGG pathway annotation using DAVID. Several predicted miR-124 target genes associated with psychiatric disorders were also identified and listed in Table S1. Some of them such as SLC6A15, MECP2 and NR3C2 have previously been reported in MDD and deregulation of these genes expression could be associated with MDD. For example, Kohli et al. found that decreased SLC6A15 expression might alter neuronal circuits related to the susceptibility for MDD (Kohli et al., 2011). Recently, Su et al. found decreased levels of MeCP2 in the peripheral blood samples of patients with MDD and in the hippocampi of depressed animals (Su et al., 2015). Likewise, Klok et al. reported that decreased expression of NR3C2 mRNA and its splice variants were detected in postmortem brain regions of patients with MDD (Klok et al., 2011). GO analysis showed that biological processes regulated by the predicted target genes included diverse terms. Some terms (e.g., neuron differentiation, neuron development, axon guidance, Wnt receptor signaling pathway, regulation of synaptic transmission, etc.) were connected in neuronal brain functions. These biological processes may be involved in the pathophysiology of MDD based on evidence from many studies (Beasley et al., 2002 and Duman, 2002). Likewise, KEGG pathway analysis revealed a close relationship between the signaling pathways identified and MDD. For instance, the axon guidance pathway, the process by which neurons send out axons to reach the correct targets, was found to play a role in neural development and function (Van Battum et al., 2015). In addition, a previous study has reported that the axon guidance pathway might participate in the pathophysiology of depression (Kao et al., 2012). Insulin signaling pathway also plays a critical role in neurogenesis and synapse transmission (Bateman and McNeill, 2006). And fluoxetine can restore the impaired hypothalamic insulin signaling pathway in animal models of depression (Pan et al., 2013). Moreover, long term potentiation (LTP) and long term depression (LTD) are also found to be implicated in depression due to their relation to synaptic plasticity (Cooke and Bliss, 2006; Gormanns et al., 2011 and Kao et al., 2012). Taken together, these biological processes and pathway analysis results illustrate possible roles of miR-124 in the pathophysiology of MDD. To date, very limited numbers of studies have investigated the effect of antidepressant treatment on miRNAs expression in MDD

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patients (Belzeaux et al., 2012; Bocchio-Chiavetto et al., 2013; Lopez et al., 2014 and Wang et al., 2015). The current study revealed a significant decrease in the miR-124 levels after eight weeks of antidepressant treatment in MDD patients, especially in responders but not in non-responders. Moreover, this study further revealed that the down-regulation of miR-124 expression was positively related to the improvement of symptoms. Taken together, these results indicate that antidepressant treatment could play a significant role in improving symptoms and simultaneously restored over-expressed miR-124 from PBMCs. Namely, miR-124 might be implicated in clinical response to antidepressant treatment in MDD patients. However, the underlying mechanism remains to be disclosed in the future. In addition, we assessed whether antidepressants (the SSRI only vs. other agents in monotherapy or in combination with SSRIs) might cause a significant difference in miR-124 levels after eight weeks of antidepressant treatment. However, we could not detect a significant difference in change in miR-124 levels of patients between the two groups (repeated measures of ANOVA, medications by time F ¼ 0.04, p ¼ 0.842). Several limitations in this preliminary study should be mentioned. First, the sample size was relatively small. Further validation in larger cohorts is necessary. Second, different types of antidepressants were used to treat patients, which could be a confounder in interpreting our findings. However, we did not find a significant difference in miR-124 levels during treatment between patients with only SSRIs and those with other agents in monotherapy or in combination with SSRIs. Third, multiple reference genes were not evaluated to determine the optimal pair of reference genes for normalization. However, the expression of U6 was demonstrated to be stable across our PBMC samples. Finally, whether the levels of miR-124 from PBMCs reflect the real levels in the CNS cannot be concluded from our study. In conclusion, in this study, we have identified that altered expression of miR-124 in PBMCs might be involved in the pathogenesis and treatment of MDD. These findings provide new information on involvement of miRNA in pathophysiological processes of MDD and in antidepressant effects. Contributors Author Shen He performed the statistical analyses and wrote the manuscript. Author Yiping Qian and Shunying Yu provided assistance for the laboratory work. Author Wu Hong completed all of the data entry. Authors Daihui Peng, Yiru Fang and Kaida Jiang were responsible for the diagnosis and clinical assessment of the participants. Author Xiaohua Liu designed and wrote the study protocol, managed the literature searches and analyses, and reviewed the manuscript. In addition, author Huafang Li offered many constructive opinions on this study and provided a critical revision of the manuscript for important intellectual content. All authors contributed to and approved the final manuscript. Role of the funding source This study was supported by projects from National Natural Science Foundation of China (81000588), “Shanghai Health System Young Talents Training Plan” of Shanghai Health Bureau (XYQ2011016), Shanghai National Science Foundation (15ZR1435400), Shanghai Key Laboratory of Psychotic Disorders (13dz2260500), National Major Project for IND, Clinical Tech Platform for Evaluation of New Drug in Psychiatry (2012ZX09303-003), Collaborative Innovation Center for Translational Medicine at Shanghai Jiao Tong University School of Medicine (TM201506) and Shanghai Health Talent Professional Project (XBR2011049).

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