Clinical And Experimental Study On Aberrantly Expressed MicroRNAs In The PBMCs Of Major Depressive Disorder

Major depressive disorder (MDD) is the most common psychiatric disorders afflicting about 5.2%～16.2% of the population worldwide. MDD has a higher rates of comorbidity with cardiovascular disease, myocardial infarction, diabetes, addiction and suicide, seriously impacting the people’s health. MDD does not only cause direct influence on patient’s quality of life, but also on their social and occupational function, imposing enormous burden on individual, family and society. Despite the great improvement on the understanding of MDD, the molecular and cellular mechanisms underlying it remains elusive.Previous research had shown that early diagnosis and Intervention of MDD significantly improve the physical, mental and social functions of patients, and greatly reduce the of treatment cost. However, so far, the diagnosis of MDD is still based on the patient’s subjective description of symptoms and on clinicians’ subjective assessment of patient’s mental status. No objective biomarker available for accurate and in time diagnosis of MDD, the chances of misdiagnosis and mistreatment are high. Thus there is an urgent need to find a biomarker with sufficient sensitivity and specificity to improve the promptness and accuracy of diagnosis.MicroRNAs (miRNAs) are a class of small non-coding RNAs about 19-25 nucleotides in length, regulating many biological processes through degradation of mRNAs or silencing their translation at the post-transcriptional level. Evidences showed that single miRNA could target up to hundreds of genes, at the same time many miRNAs could regulate the expression of one gene, which made miRNAs the fine-tuner and the key node of the gene expression and the signal pathway network. More than 50% of the human protein-coding genes were regulated by miRNAs. A large portion of miRNAs are expressed specifically in the central nervous system and play an important role in the brain development, neurogenesis, neural maturation, and synapse formation. Further studies showed that the alterations in miRNA expression might contribute to the onset of neuropsychiatric disorders such as schizophrenia, autism, and mood disorder. Several lines of evidence showed that miRNAs have participated in the pathomechanism of MDD:1. miRNA polymorphisms play an important role in MDD susceptibility; 2. miRNAs affect the development of MDD through regulate the neurogenesis; 3. miRNAs affect the development of MDD through regulate the and synaptic plasticity; 4. Stress is a important risk factor of MDD, while stress could change the miRNAs expression profiles in the brains of the animal models of depression.5. The expression of miR-132 could be upregulated by BDNF and then affects the neurons neurite formation, while it had been proven that BDNF has a close relation with MDD.6. The expression of miRNAs could be influenced by antidepressant both in animal models and cultured cells.As a biomarker, it should be stable, reproducible, and easily accessible, while peripheral miRNA just possess all those qualities. Peripheral miRNAs have stable physicochemical property, resistant to nuclease degradation; They can be detected by PCR with high accuracy and repeatability; The expression of miRNAs is specific to tissues or biological stages; The detection of miRNAs is easily accessible, non-invasive, inexpensive. More importantly, miRNAs in brain tissue and pripheral blood share a common miRNA expression pattern; In addition, the expression profiling of miRNAs in lymphocyte correlates closely with the clinical symptoms, suggesting the metabolism of blood cells might reflect that of brain cells. So we wonder whether peripheral miRNAs can serve as a biomarker of MDD?Based on the above-mentioned research background, a study was designed to find miRNAs which might serve as biomarkers for MDD, and to explore the role of miRNAs in the pathomechanism of MDD. This study divided into four parts, as described below.1. Differential expression of microRNA in peripheral blood mononuclear cells of major depressive disorderMethods:Total RNAs from three MDD patients and three controls were used for miRNA microarray profiling. The expression of miRNAs in peripheral blood mononuclear cells (PBMCs) was measured by Affymetrix miRNA 3.0 array.10 miRNAs were chosen for further validation with qRT-PCR in a larger sample of 91 cases and 46 controls. Finally ROC curve analysis was conducted to estimate the sensitivity and specificity of the differentially expressed miRNAs signature.Results:1.26 miRNAs were found differentially expressed through miRNAs microarray analysis, among which 21 miRNAs were up-regulated and 5 others were down-regulated.2. PCR validation showed that 5 miRNAs (miR-1972、miR-26b、 miR-4485、miR-4498、miR-4743) up-regulated with statistical significance(P<0.05).3. Receiver operating characteristic (ROC) curve analysis showed that the combining area under the ROC curve (AUC) of these five miRNAs was 0.636 with 54.35% sensitivity and 79.01% specificity.2. The effect of antidepressants on the expression of miRNAs in PBMCs of MDDMethods:27 patients with MDD received oral selective serotonin reuptake inhibitors (SSRIs) treatment for 3 weeks. miRNAs expression levels were detected by qRT-PCR and clinical symptoms were assessed by using Hamilton Depression Scale (HAMD) before and after 3-week treatment.Results:1. Total score and all the factor scores of HAMD except "diurnal variation" were significantly decreased after medication(P<0.01).2. The expression levels of miR-1972, miR-26b, miR-4485, miR-4498, and miR-4743 were significantly down-regulated after medication (P< 0.05 or P< 0.01).3. The changes of expression level of miR-26b was positively correlated with the change of HAMD total score, the factor scores of anxiety/somatization and cognitive impairment (P<0.05); The changes of expression level of of miR-1972 was positively correlated with the change of HAMD total score and the factor score of cognitive impairment (P<0.05); The changes of expression level of miR-4743 was positively correlated with the change of factor score of cognitive impairment (P<0.05).3. Bioinformatics analysis of microRNAs differently expressed in major depressive disorderMethods:The targets genes of the five miRNAs (miR-26b, miR-1972, miR-4485, miR-4498, miR-4743) were predicted by Target Scan, miRBD, and DIANA-microT-CDS, and the genes coidentified by all three programs were considered as target genes of a given miRNA. Then FunNet (http://www.funnet.ws/) was used with default settings for GO terms and KEGG pathways enrichment analysis.Results:1. GO biological process analysis showed that the target genes of the 5 miRNAs have been involved in many biological processes, among which several important biological processes played a significant role in brain development and function.2. KEGG pathway analysis showed significant enrichments in several pathways related to neuronal brain function, such as axon guidance, glutamatergic synapse, Wnt signaling pathway, ErbB signaling pathway, mTOR signaling pathway, VEGF signaling pathway, long-term potentiation etc, which may be relevant to the pathophysiology of MDD.3. miR-26b, miR-1972, and miR-4498 lies at the central of the miRNA-gene network, miRNA-biological process network, and miRNA-pathway network, suggesting these 3 miRNAs may have more important regulatory functions.4. Association study of miR-26b and mTOR signal pathway in mice model of depression induced by neonatal maternal separationMethods:Tow pregnant mice were assigned to Neonatal maternal separation (NMS) and normal control (NC) group randomly. After parturition, NMS was conducted to induce depression model. At 60 days post-partum the pups’depression-like behaviors were assessed by the forced swimming test and tail suspension test. Then 6 NMS mice and 6 normal control (NC) mice were killed and the expression level of miR-26b in the frontal cortex was measured, as well as the content of mTOR, p-mTOR, Akt, and p-Akt. Then the spearman correlation analysis was conducted to evaluate the correlations between expression of miR-26b and the ratios of p-mTOR/mTOR and p-Akt/Akt.Results:1. Phosphorylated (p)-mTOR/mTOR and p-Akt/Akt ratios in the frontal cortex of the NMS were significantly decreased compared to the NC (P<0.05 or P<0.01). 2. The relative expression level of miR-26b in the frontal cortex of the NMS was significantly increased than that of the NC (P<0.01).3. The relative expression level of miR-26b was negatively correlated with the p-mTOR/mTOR ratio (P<0.05).Conclusions:1. There were differential expression of miR-26b, miR-1972, miR-4485, miR-4498, and miR-4743 in PBMCs of patients with MDD. The differentially expressed miRNAs were sensitive to antidepressant treatment, and their expression level correlated with the clinical symptoms. miRNAs in PBMCs have the potential to serve as biomarkers for MDD.2. Bioinformatics analysis showed significant enrichments in several GO biological processes and KEGG pathways which are relevant to the neuronal brain function and the pathophysiology of MDD. miR-26b, miR-1972, and miR-4498 may have more important regulatory functions.3. NMS can induce increased expression of miR-26b as well as decreased ratios of p-mTOR/mTOR and p-Akt/Akt in frontal cortex of NMS mice. The expression level of miR-26b was negatively correlated with the activity of mTOR signaling pathway in frontal cortex of NMS mice.