Clinical And Experimental Study On Aberrantly Expressed LncRNAs In The PBMCs Of Schizophrenia

Schizophrenia(SZ) is one of the most popular serious psychiatric diseases afflicting about 1% of the population worldwide. The clinical features of SZ mainly includes the positive symptoms such as hallucinations, delusions, psychomotor excitement and(or) with emotional slow, emotional indifference, social withdrawal, will decline, such as negative symptoms. The illness cause patients persinality change,emotion and behavior decline, social adaptation ability drop and social work badly damaged. Majority of those affected will develop chronicity with frequent relapse and admission, which does not only cause serious influence on patient’s social and occupational function, but also on their quality of life, enhancing enormous burden on the family and society. Despite progress have made in the last decades, the underlying pathophysiology of schizophrenia remains not well understood.Current evidences demonstrate that SZ is attributable to the interactions between environmental and genetic factors. However, till today, the diagnosis of SZ is still based on clinical symptoms varying greatly between individuals and on the patient’s subjective description of symptoms. There is no objective biomarker in the diagnosis of SZ, which may lead to misdiagnosis or different diagnosis between doctors. So it is urgent to find a objective and feasible clinical diagnostic index with accurate sensitivity and specificity to elevate the quality of early diagnosis for SZ.Long non-coding RNAs(lncRNAs) are a class of transcripts non-coding RNAs longer than 200 nucleotides and lacking an appreciable open reading frame, which do not encode for protein. Although initially thought to be transcriptional noise, lncRNAs have been shown to be involved in major mechanisms of gene expression regulation and cellular development. Lnc RNAs engage in post-transcriptional regulation of target RNAs such as: protein synthesis, maturity and transshipmen of RNAs. Through chromatin modifications or silencing their translation, lncRNAs can aslo modify many biological processes at the post-transcriptional level. A growing evidence has demonstrated that lncRNAs participate in diverse biological processes by regulating gene expression, cell proliferation and differentiation, which made lncRNAs a key role in the of the gene expression network and signal transduction pathway. A large portion of lncRNAs which highly expressed in brain also be found altered specifically in neuropsychiatric disorders, which indicated that lncRNAs play an important role in the cerebral development, cell proliferation, neurophysiologic maturation, and the occurrence of psychiatric illness. However, the lncRNA expression profiling in SZ patients is little reported. Until now, only a few studies reported that lncRNAs may play important roles in various pathologic processes in SZ.As a good biomarker, it should be reproducible, stable and easy to access, while peripheral lncRNAs just have these advantages. Peripheral lncRNAs have stable physical and chemical properties, stand up to the degradation of nuclease; With high measurement repeatability and accuracy tested by PCR, the detection of lncRNAs is easy to access, non-invasive and cost-effective; The level of lncRNAs expressed a scheduling and the height of the tissues specificity or biological stages; Moreover, lncRNAs in brain tissue and pripheral blood leulocytes have many common biological pathways and many similar gene expression; In addition, the aberrant expression of lncRNAs in PBMCs spectum are related closely to the clinical manifestations, suggestting the metabolism of peripheral blood lymphocyte cells might reflect that of the neurocyte. Therefore, finding related lncRNAs as biomarkers of SZ is our research topics.On the basic of the above background, We tried to find aberrant lncRNAs which might serve as biomarkers for the diagnosis of SZ, and to explore the role of the finding dys-regulated lncRNAs in the pathomechanism of SZ. This study described below including four parts.1. Differential expression of lncRNA in peripheral blood mononuclear cells of Schizophrenia Methods: Using lncRNA microarray profiling, total RNAs were extracted from 3 SZ patients and 3 controls. The lncRNAs in PBMCs were screened and compared between the SZ patients and controls using Agilent Human lncRNA microarray. Using q RT-PCR, 10 top changed lncRNAs were selected for further validation in a larger sample of 106 cases and 48 controls. Finally, the sensitivity and specificity of the differentially expressed lncRNAs signature was estimated by ROC curve analysis. Results: 1. One hundred and twenty-five lncRNAs were significantly differentially expressed in SZ patients compared with healthy controls, of which 62 were up-regulated and 63 were down-regulated. 2. PCR validation indicated that NONHSAT089447, NONHSAT021545 and NONHSAT041499 exhibited significant difference of expression between patients and healthy controls(P < 0.05) 3. Receiver operating characteristic(ROC) curve analysis demonstrated that the combining area under the ROC curve(AUC) of the above three lncRNAs was 0.791 with 62.5% sensitivity and 75.0% specificity.2. The effecacy of antipsychotic treatment on the expression of lncRNAs in PBMCs of SZ Methods: Three verified significantly dys-regulated lncRNAs(NONHSAT089447, NONHSAT021545, NONHSAT041499) of PBMCs were selected and then measured in 30 SZ patients before and after the antipsychotic treatment. SZ symptomatology improvement was estimated by Positive And Negative Syndrome Scale(PANSS) scores. Results: 1. Total score and all the factor scores of PANSS were significantly decreased after medication for 8 weeks(P < 0.01). 2. The ΔCT values of lncRNAs NONHSAT089447 and NONHSAT041499 were significantly increased in patients after treatment for 8 weeks(P<0.001), indicating the significant down-regulation of these lncRNA expression by the treatment. 3. Pearson correlation analysis revealed that the down-regulation of the lncRNA NONHSAT041499 expression was significantly correlated with the improvement of positive and activity symptoms after the treatment(r=-0.444 and-0.423, respectively, P < 0.05).3. Bioinformatics analysis of the dys-regulated lncRNAs differently expressed in SZ Methods: The co-expressed m RNAs of lncRNAs were identified by calculating Pearson correlation and the result were analyzed by gene ontology and KEGG pathway analysis using DAVID. Then a lncRNA-transcription factor-gene network was performed. Results: 1.Totally 89 co-expressed m RNAs were identified of the three lncRNAs(NONHSAT089447 、 NONHSAT021545 、 NONHSAT041499), among which several crucial GO biological processes played an important role in central nervous system development and function. 2. KEGG pathway analysis showed the targets genes of the co-expressed m RNAs significant enrichments in several pathways related to the pathophysiology of SZ. 3. NONHSAT089447、NONHSAT021545 and NONHSAT041499 lies at the central of the m RNA-gene network and m RNA-pathway network, indicating these 3 lncRNAs may have be a crucial regulators to the pathophysiology of SZ.4. Study on the regulatory mechanism between differentially expressed lncRNA(NONHSAT089447)and dopamine signaling pathway in schizophrenic patients Methods: During culturing human neuroblastoma cell lines(SK-N-SH), dopamine(DR) was added to simulate nerve cells in schizophrenic patients, then the expression level of lncRNAs were detected by real-time fluorescence quantification PCR.On the opposite side, olanzapine was used to prove the increase of NONHSAT089447 was caused by DR. si RNA and plasmid-447 were transferred into SK-N-SH by lipofectamine transfection, after culturing, extract total RNAs, then the expression levels of two lncRNAs(NONHSAT089447,NONHSAT041499), as well as the expression change of dopamine receptors(DRD1, DRD2, DRD3, DRD4, DRD5) before and after transfection were detected by q RT-PCR. Western blot was applied to detect the change of DR downstream signaling after the interference and over expression of NONHSAT089447. Results: 1. q RT-PCR suggested addition of OLP suppressed the expression of NONHSAT089447 significantly; 2. si RNA showed more suppressing effect on the expression of DRD3 and DRD5(P<0.05); 3. NONHSAT089447 overexpression improved the expression level of DRD3 and DRD5(P<0.05).4. Western blot suggested interference and overexpression of NONHSAT089447 made DRD downstream signaling declined and enhanced respectively. Conclusions: 1. There were differential expression of NONHSAT089447 、NONHSAT021545 and NONHSAT041499 in PBMCs of SZ patients. The dys-regulated expressed NONHSAT089447 and NONHSAT041499 were changed by antipsychotic treatment, and the level of change correlated with the clinical manifestation. The 3 aberrant lncRNAs in PBMCs might be involed in the pathogenesis and the development of SZ.2. Bioinformatics analysis indicated 89 co-expressed m RNAs were identified of the three lncRNAs(NONHSAT089447 、 NONHSAT021545 、 NONHSAT041499), and KEGG pathways showed the targets genes of the co-expressed m RNAs are relevant to the central nervous system function and the pathophysiology of SZ. NONHSAT089447 、NONHSAT021545 and NONHSAT041499 may have more important regulatory functions in SZ. 3. NONHSAT089447 expressed highly in SZ patients and the dopamine signaling pathway is stimulated by high-expressed NONHSAT089447 to accelerate the expression of NONHSAT089447 again, which is a positive feedback loop. NONHSAT089447 in PBMCs have the potential to serve as biomarkers for SZ. The expression level of it may be a useful standard in evaluating SZ therapy.