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

Depression is a comparatively common mental disorder,characterized by enduring sadness,anhedonia,feelings of guilt or low self-worth,disturbed sleep or appetite,feelings of tiredness,and poor concentration and even suicidal thoughts.Up to 2012,more than 350 million people suffer from depression all over the world,patients suffering from Depression?MDD?will get a 12-month prevalence of 6.6% and a lifetime prevalence of 16.2%,and women are twice susceptible compared to men.Compared to unipolar depression patients,bipolar patients are much more likely to present to clinicians,while unipolar depression is more prevalent than bipolar disorder.The clinical presentation of patients with unipolar depression is more likely to show typical depressive symptoms including sad mood,anxiety,insomnia,and somatic depressive symptoms.However the clinical presentation of patients with bipolar disorder,are more likely to show “inverse” neuro-vegetative symptoms,particularly hypersomnia,increased appetite and weight gain.Current antidepressants,such as serotonin?5-HT?re-uptake inhibitors?SSRIs?were commonly applied in treating depression.Recent years,advances have been made in improving the diagnosis and treatment of MDD patients,for example,many promising biomarkers have been intensively investigated for the early diagnosis of MDD and as treatment targets for novel drug development.So in the future study,we still need a lot of effort to achieve these new diagnostic and treatment methods.According to the research on the human genome,though only less than 1% of the mammalian genome that are transcribed into m RNA,the major part of the genome is non-coding RNAs?nc RNA?what was previously considered as “transcriptional noise”,that do not encode information about proteins.Long non-coding RNAs?lnc RNAs?are those over 200 nucleotides in length and do not have a functional open reading frame.Lnc RNAs have multiple functions including the ability to regulate transcription/ gene expression by recruitment of transcription factors,modulate m RNA processing,adjust post-transcriptional pathways?like translation,mi RNA sponges and m RNA stability?,DNA methylation and scaffold to organize and hold higher-order complexes,modulate chromatin structure by recruitment of histone and chromatin modifiers to specific genomic loci and so on.Thus,lnc RNAs may play some roles in disease pathologic process,and intense investigations have been implemented to find new molecules and mechanisms of complex diseases,providing light on the potential of utilizing lnc RNAs as the diagnosis,prognosis,and also promising treatment targets.Accumulating evidence showed that lnc RNAs with a large number and diversity play an important regulator role in brain functions.Lnc RNAs are highly expressed in the central nervous system,affecting neural stem cell maintenance,neurogenesis and gliogenesis,brain patterning,synaptic and stress responses,neural plasticity and cognitive function.For example,lnc RNA brain cytoplasmic?BC1?is one of the first lnc RNA characterized in the brain,is now known to regulate metabotropic receptor signaling.Neurogenesis-associated lnc RNAs were found to associate directly with REST,the transcription factor SOX2 and PRC2 component SUZ12,suggesting that lnc RNAs may act as guides for these proteins.Importantly,knockdown of these lnc RNAs by RNAi resulted in impaired neuronal differentiation,suggesting that lnc RNAs are critical regulators of neurogenesis.Therefore,it provides a new perspective to determinate whether the development of MDD has certain underlying association with lnc RNA,and if the hypothesis were plausible,what kind of detailed mechanisms involved in the process.Part ? Screening and Verification of Abnormal Expression of lnc RNA in PBMCs of DepressionAIM Human lnc RNA microarray profile was used to screen the abnormal expression of lnc RNAs and RT-PCR was used to verify lnc RNAs in PBMCs of depressive patients.METHOD Three patients with depression who met the diagnostic criteria of DSM-IV and three healthy controls were selected.Extraction of peripheral venous blood,extraction of mononuclear cells,separation and extraction of RNA,with human lnc RNA 3.0 chip?including 40,173 human lnc RNAs?were selected to screen out the differential expressed lnc RNAs.Ten candidate lnc RNAs were then identified by q RT-PCR in 138 patients with depression and 63 healthy controls,and the selection criteria were: in the results of cluster analysis,lnc RNAs were selected in different clusters;there were a large variation factor?FC?in the results of the chip screening;lnc RNAs were expressed in all the subjects.SPSS was used to analyze the differential expression of lnc RNAs in subjects by operating characteristic curve?Okazaki et al.?.RESULTS After screening by the chip,2649 lnc RNAs were found to be differentially expressed in depressive patients,with 534 significantly up-regulated and 2115 significantly down-regulated.Ten lnc RNAs?ENST00000414201,ENST00000591189,TCONS_L2₀0001212,NONHSAT102891,TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,and NONHSAT142707?were identified in the large sample?138 in the MDD group and 63 in the NC group?.Eight lnc RNAs?TCONS_L2₀0001212,NONHSAT102891,TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,NONHSAT142707?were significantly different compared to the healthy control?P<0.05?,and they showed a downward trend.ROC curve analysis showed that the area under the curve?AUC?of the eight lnc RNAs was 0.596-0.646,the area under the ROC curve was 0.719,the sensitivity was 82.4%,and the specificity was 72.0%.CONCLUSION Compared with the healthy control group,the eight significant down-regulated lnc RNAs?TCONS_L2₀0001212,NONHSAT102891,TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,and NONHSAT142707?were differentially expressed in PBMCs of depressive patients and could be considered as having the potential to diagnose biomarkers as Depression.Part ? Effect of antidepressant drugs on lnc RNAs expression in PBMCsAIM To explore whether the expression of eight down-regulated lnc RNAs?TCONS_L2₀0001212,NONHSAT102891,TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,and NONHSAT142707?could change with depressive symptoms in depressive patients after treatment with normal antidepressant drugs,and bioinformatics analysis of lnc RNAs with significant changes was made.METHOD A total of 30 patients with depressive drugs who were treated with antidepressants?S-citalopram,mirtazapine,sertraline and fluoxetine?were randomly selected for 3 weeks and 6 weeks treatment,and PBMCs were isolated and extracted,then the Hamilton Depression Scale 24 item?HAMD24?was used to assess the current state of depression,q RT-PCR was used to detect the expression of eight lnc RNAs in the first part of the depression in 30 patients and compared with the healthy control group.GO was used to analysis the biological process of differential lnc RNAs and KEGG was used to analysis the signal pathway enrichment.RESULTS After 6 weeks treatment,the HAMD scores of 30 patients with depression were significantly lower than before treatment?P<0.05?.Among them,24 patients achieved clinical curve and 6 patients achieved responsive to drug therapy.After variance analysis of the repeated measurement design,the expression of six lnc RNAs?TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,NONHSAT142707?were significantly up-regulated at 6 weeks?P<0.05?,and there was no significant difference between the control group?P>0.05?.GO biological enrichment analysis showed that m RNA co-expressed with lnc RNAs involved a variety of biological processes,including translation extension,protein transport,protein localization,protein complex biological origin,protein complex establishment,translation,macromolecule complex assembly,proteasome protein catabolism process,etc;the results of enrichment analysis of KEGG signaling pathway suggest that lnc RNAs contain ribosomes,Alzheimer's disease,RNA degradation,pancreatic cancer,Parkinson's disease,cell cycle,DNA ‘s replication,prostate cancer,Huntington's disease,long-term depression,etc.CONCLUSION The six significant down-regulated lnc RNAs?TCONS₀0019174,ENST00000566208,NONHSAG045500,ENST00000517573,NONHSAT034045,and NONHSAT142707?can be used as clinical practice,the potential diagnostic criteria for depression and treatment markers.Part ? Cross-validation of Differential Expression of lnc RNAs in Depression,Schizophrenia and Anxiety DisorderAIM Depression or anxiety are apparent symptoms in the early onset or acute phase of schizophrenia?Rinn et al.?,which complicates timely diagnosis and treatment.It is imperative to seek an indicator to distinguish early onset or acute schizophrenia from depressive or anxiety disorders.This study examines the specific diagnostic value of the six lnc RNAs above in the diagnosis of depression and schizophrenia and anxiety,which is the specific expression of lnc RNAs in depression.METHOD Using the lnc RNA microarray profiling and RT-PCR technique,three significant up-regulated schizophrenia lnc RNAs,six down-regulated lnc RNAs and three significant up-regulated anxiety lnc RNAs which were found in this panel were cross-validated in 40 Schizophrenia patients,40 depression patients,40 anxiety patients and 40 healthy controls.RESULTS Six down-regulated depression lnc RNAs was significantly up-regulated in schizophrenia,and there were no significant difference of expression trend and severity than in anxiety;three up-regulated schizophrenia lnc RNAs and three up-regulated anxiety lnc RNAs were significantly down-regulated in depression.CONCLUSION The expression of six significant down-regulated lnc RNAs was specific in depression and could be used to identify depression with schizophrenia and anxiety.Part ? Study on Regulatory Mechanism of lnc RNAs and Serotonin Signaling Pathway in DepressionAIM Depression?MDD?is a chronic,life threatening and highly disabling disease.Standardizing treatment with less side effect,high efficiency of short treatment time,quick effect,and long maintenance for MDD is always being pursed.Long noncoding RNAs?lnc RNAs?are highly expressed in the central nervous system,affecting neural stem cell maintenance,brain patterning,synaptic and stress responses,neural plasticity and cognitive function,and proved to be involved in the development of neurodegenerative and psychiatric diseases.This study aimed to investigate the association between the over-expression and interfering of three differentially down-regulated lnc RNAs?NONHSAT142707,NONHSAG045500,and ENST00000517573?in MDD and central neurotransmitter 5-hydroxytryptamine transporter SERT in SK-N-SH cell.METHOD We firstly synthesized and validated the effect of three lnc RNAs plasmid and small interfering RNAs?si RNAs?,then transfected the plasmid and si RNAs which had significant over-expression or interfering effect to SK-N-SH cells and tested the expression of SERT in SK-N-SH cells by q RT-PCR.RESULTS The expression of the three lnc RNAs plasmid had significant over-expression effect;each lnc RNAs si RNA1 has sifnificant interfering effect,while si RNA2 has no such significant effect.Only the overexpression of NONHSAG045500 could significantly inhibit the expression of SERT,meanwhile,the interfering of NONHSAG045500 could significantly strength the expression of SERT.CONCLUSION This study indicated that the expression of SERT in SK-N-SH cell in vitro could be regulated by up-regulating or down-regulating NONHSAG045500,and suggested that NONHSAG045500 seems to be established as a new therapeutic target of MDD.Future work may be needed to definitely determine the correlation between NONHSAG045500 and SERT in vivo.