A Preliminary Study On Functions, Mechanisms Of Human EBLN1Gene And Its Role In Major Depressive Disorder

BACKGROUDRecent studies suggest many viruses can become part of the geneticmaterial of their host species, a process that is called endogenization. Suchendogenous viral elements (EVEs) result from the chromosomal integrationof viral DNA (or DNA copies of viral RNA) in the host germ cells. Althoughendogenization was for a long time thought to be limited to retroviruses,more surprising was the recent discovery that eukaryotic genomes containsequences from RNA viruses. Similarly, several single stranded DNAviruses have left integrated copies in their host genomes. Above three typesof EVEs are designated Endogenous Retrovirus (ERV), Endogenous DNAVirus-like Elements and Non-retroviral Integrated RNA Virus (NIRV)respectively. Their establishment, evolution and potential function haveattracted considerable interest.Horie et al. reported that non-retroviral viruses called Borna diseasevirus (BDV) have been endogenized repeatedly during mammalian evolution including humans, which were designated endogenous borna-likeN (EBLN) elements. It was first report that endogenous non-retroviralelement was found in the genomes of mammalian species. There are fourEBLNs in human genome designated EBLN1, EBLN2, EBLN3, EBLN4, inwhich showed the highest overall58%sequence similarity to BDV N.EBLN1has an ORF encoding366amino acids, which is comparable withthe full-length BDV N protein (370amino acids). The ORF is also conservedamong other haplorhini primate species and EBLN1nucleotide sequencehave been detected in the human EST database. In addition, it is found thatEBLN1could express as mRNA in several human cell lines, e.g. OL,293T,MOLT4. These data suggested that EBLN1encode functional protein.However, as a novel identified NIRV gene, it is still unknown for its structure,systematic expression profile, specific functions and role in certain diseases.BDV is a typical and well studied virus which probably involved inparticular psychiatric diseases. BDV is a non-cytolytic, non-segmentedRNA virus of negative polarity, and is the only member of the familyBornaviridae within the order Mononegavirales. In addition, the entire lifecycle of BDV takes place in the nucleus of the infected cells. Recent findingsfrom numerous epidemiological surveys, case control studies, virus isolationexperiments and clinical trials suggest that BDV infection is correlated withpsychiatric diseases including Major depression and bipolar disorder, andindicate BDV infection may be a human mental-health risk. BDV targets mainly neurons of the limbic system and persists primarily in thehippocampus. In rats, BDV causes disturbances in learning, mood, andbehavior reminiscent of symptoms observed in human psychiatric diseasessuch as mood disorders, schizophrenia and autism. Based on close linkbetween BDV infection and psychiatric diseases, BDV was proposed as“mood” virus at the International Berlin Symposium on BornavirusInfections in2008.HERVs, belonging to EVE the same as NIRVs, was discovered in early1970s, and has been well studied. Genome sequencing reveals that8%of thehuman genome consists of HERVs.Many reports suggested that HERV could encode proteins involvingdevelopment of the placenta, immunologic suppression, cell differentiation,regulate expression of other genes as cis-acting element and antivirusimmunity. In addition, abnormal expression of HERVs probably is linkedwith particular human diseases, such as autoimmune diseases, schizophreniaand cancers.To sum up, EBLN1showed overall58%sequence similarity to BDV N,which may keep similar biological feature and pathogenicity. Since EBLNsand HERVs are both belonging to EVE and molecular fossils in host genome,we could speculate that EBLNs own similar biological feature and abnormalexpression of EBLNs probably is linked with particular human diseases asHERVs. In addition, it was evidenced that EBLN1could express as mRNA in OL cell line, which is a kind of human neural cell line. Based on above all,we hypothesize that EBLN1may have some vital fuctions and involve indevelopment of depression, which act as a bridge linking BDV infection anddepression.OBJECTIVEWe aim to discover specific functions, mechanisms and role indepression for EBLN1, idenfity whether it is a novel predisposing gene, andprovide molecular genetics evidence for association between BDV infectionand MDD. Then provide basis for further understanding to this novelidentified NIRV gene, and also provide some clues for further elucidatingBDV infection and depression.METHODS1. Systematic expression profile of EBLN1was obtained by RT-PCR inSY5Y, U87, OL,293T, PBMC. After that total RNA was extracted from OLcell line. Then full cDNA sequence was cloned by5’-RACE and3’-RACEapproachs and the open reading frame (ORF), untranslated regions (UTR)and encoding amino acids were predicted.2. EBLN1RNAi lentivirus vector was constructed and packaged intovirus particle. Two BDV strains were used to infect EBLN1RNAi group andnegative control OL cells repectively. RT-qPCR, WB, FFU were employedto detect BDV p24/p40genes, proteins and titre, in order to evaluate effect ofEBLN1on OL cell susceptibility to two BDV strains. In addition, proliferation, migration and motion abilities were compared betweenEBLN1RNAi group and negative control OL cells by MTT, Transwell andWound healing assays. Moreover, DNA microarray was performed toanalyze downstream changing gene prolile of EBLN1.3. Total94PBMCs samples were collected from MDD patients andhealthy controls respectively.Total RNA was extracted and relativetranscript levels of EBLN1were compared between two groups by RT-qPCR,normalized by GAPDH, in order to clarify the correlation of EBLN1transcription level and MDD.4. Total389MDD patients and301healthy controls were enrolled inthis study. Genomic DNAs were extracted from200μl whole blood, andthen were subjected to PCR sequencing, aiming to screen SNP sitesofEBLN1in Chongqing population and conduct SNP association analysis.RESULTS1. EBLN1transcripts were detected in all five selected human cell typeby RT-PCR. The full cDNA sequence of EBLN1gene was obtained byRACE approach, which is longer than it in Genbank and contains a5’UTRsequence actually2. Two BDV strains p24/p40genes, proteins and titre in EBLN1RNAigroup were significantly higher than negative control OL cells at mosttimepoints, and results from RT-qPCR, WB, FFU showed consistenttendency. In addition, MTT results showed proliferation ability in EBLN1 RNAi group decreased than negative control OL cells on2ndDay to5thday（P<0.05）. Transwell results showed migration ability in EBLN1RNAigroup decreased than negative control OL cells at20thhour（P<0.05）.Wound healing assays results showed motion ability in EBLN1RNAi groupdecreased than negative control OL cells at6thand20thhour（P<0.05）.Moreover, from DNA microarray,1067up-regulated and2005down-regulated genes were found. Pathway analysis indicated EBLN1couldinduce multiple signal pathways alternation, and dominant pathways focuson cell cycle, MAPK, P53and apoptosis.3. It was showed that relative transcript level of EBLN1in MDD group(1/4of control) were significantly lower compared healthy controls（P<0.001）.4. Based on PCR sequencing results, no SNP sites of EBLN1werefound in Chongqing population investigated and no evidence was found tosupport the association between EBLN1polymorphism and susceptibility ofdepression.CONCLUSION1. It is evidenced that EBLN1express more widely in human tissue thanpreviously thought. Especially, EBLN1expresses highly in three types ofneural cell lines, which indicates that it play a vital role in nervous systemphysiology. The full cDNA sequence of EBLN1gene was obtained byRACE approach, that provides basis for further research on functions of EBLN1and role in diseases.2. EBLN1owns functions of antivirus immunity against exogenousBDV, and promoting proliferation, migration and motion abilities in OL cellline. Through DNA microarray we speculate that MAPK, P53, cell cyclepathways or RND3gene involved in proliferation promotion of EBLN1.These results are the preliminary discovery for functions and mechanism ofEBLN1.3. We evidence that relative transcript level of EBLN1in MDD patientsdecreases, predicting it has a anti-depressive role.4. No SNP sites of EBLN1were found in Chongqing populationinvestigated and no evidence was found to support the association betweenEBLN1polymorphism and susceptibility of depression, which suggest thedecrease of EBLN1transcription in MDD patients is not due topolymorphism.