Phylogenetic Analysis And Biological Function Of Human EBLN1 Gene

BACKGROUNDHuman endogenous retroviruses(HERVs)is generated by integration of viral DNA into the human genome during viral infection and inheritance to next generation.About 8% of human genome is composed of HERVs.HERVs wre involved many normal physiological functions,such as embryonic development and immune regulation.At the molecular level,HERVs may participate in regulation of gene expression through a variety of ways,such as promoters,enhancers or silencers,polyadenylation signal,and nuclear protein binding sites.Studies have shown that abnormal expression of HERVs is closely related to certain human diseases,including neurological and psychiatric disorders,cancer and autoimmune diseases.For example,HERV-W1 was highly expressed and aberrantly activated in many cancers.Thus,in-depth study of the biological function of HERVs is conducive to reveal the pathogenesis of human disease.HERVs is expected to become a new target for therapy.EBLN(endogenous borna-like N elements)has a high identity with and the nucleoprotein(P40)of borna disease virus(BDV),which is a non-retroviral virus.So far,seven EBLNs have been found in human genome,named EBLN1~ EBLN7,amongst of which EBLN1 has the most homology to the amino acid sequence of BDV N protein.However,the biological functions EBLN1 have poorly been understood.It has been confirmed that EBLN1 was not only highly expressed in 293 T cells and MOLT4 cells,but also in neurological tumor cells,such as oligodendrocytes(OL)cells,neuroblastoma cells(SY5Y),and human glioblastoma cells(U87).It is suggested that EBLN1 may play an important role in maintaining the malignant phenotype of nerve tumor.BDV is a single negative-strand RNA virus,contains six open reading frames(open reading frame,ORF)and expresses at least six viral proteins(N,P,X,L,G and M).The protein N(P40)and P(P23)are key factors for BDV disease.BDV is highly neurotropic,and mainly affects the limbic system.Animal experiments show that BDV infection can cause the host to have abnormal behavior,learning and memory disorders,and mood disorders.Epidemiological surveys showed that the BDV infection rate in patients with schizophrenia,bipolar disorder,and other neuropsychiatric disorders was significantly higher than the control group.BDV may be a etiological factor of human neuropsychiatric diseases.It is reasoned that the biological function EBLN1 may also be related to the nervous system.OBJECTIVE1.To sequence the whole genome of the five BDV strains(Hu-H2,HUSA1,Dessau,Cresalo and WR3),and to analyze the phylogenetic relationship between EBLN1 and human-derived BDV N gene.2.To investigate the roles of EBLN1 in nerve tumors.3.To explore biological functions of EBLN1 protein in nervous system.METHODS1.Amplification the whole genome of the five BDV strains by using RT-PCR.The whole genome sequences of the five BDV strains and the reference sequences obtained from GenBank was compared by using DNAMAN software.Then the evolutionary tree was constructed by using MEGA software.By the same way,EBLN1 and the BDV N gene were compared.2.EBLN1 was inhibited by a RNAi lentiviral vector.After infection,cell proliferation,apoptosis,cell cycle,colony formation assays,Would scratches and Transwell migration assay were performed to test the effects of EBLN1 silencing on the biology characters of OL,SY5 Y,and U87 cells.The level of mRNA expression of genes was detected by qRT-PCR.The proteins relative to cell cycle and apoptosis were detected by western blotting.3.The lentiviral vectors and adeno-associated virus vector for EBLN1 expression were used to infect hippocampal neurons and to inject into rat hippocampus,respectivrly.After infection,immunofluorescence was used to detect EBLN1 cellular localization.The gene expression profiling was measured by microarray.The proteins related to synaptic plasticity were tested by western blotting.After injection,Morris water maze was performed to evaluate the ability of learning and memory in rats.RESULTS1.Amplification and sequencing of the whole genome of five BDV strains from different hosts(Hu-H2,HUSA1,Dessau,Cressalo and WR3)were completed.Further analysis showed that the identity between human-derived and non-human-derived BDV was 98.07%~99.97%.BDV strains had a geographical features.In addition,the identity between EBLN1 and human-derived BDV N gene was 47.6% ~ 50.82%.EBLN1 had a closer genetic relationship with the BDV N gene derived from a German patient.2.EBLN1 interference could alter the biological characters of OL,SY5 Y,and U87 cells.EBLN1 silencing in OL cells could inhibit proliferation,increased apoptosis,and reduce colony formation.EBLN1 silencing in SY5 Y cell could reduce the ability of movement and migration.EBLN1 silencing U87 cell could inhibit proliferation,increased apoptosis,and reduce colony formation,and reduce the ability of movement and migratory.EBLN1 interference could induce the higher expression of RND3 OSMR and CREB3L2,which are closely related to the proliferation and apoptosis in glioblastoma.Meawhlie,the expression of Bcl-2,an apoptosis inhibitory protein,was significantly decreased.The expressions of Bax,caspase 3 and P53 had no significant difference after EBLN1 interference.3.EBLN1 protein is localized in the cytoplasm.After expression of EBLN1 protein,a toal of 170 genes were dysregulated including 10 upregulated genes,such as NEFM,CCDC148,GPR85,and 160 downregulated genes,such as DES,ABCD5,GOLGA6 C,ANXA2,PARP4 like.GO analysis showed that the functions of differential genes focused on cell cycle,RNA transcription regulation,chromosome condensation and separation.KEGG analysis found that the defferential genes were mainly invovled in the ribosomal signaling pathway.Meanwhile,EBLN1 protein could increase the expressions of PSD95 and pERK.However,the results of Morris water maze showed that EBLN1 protein did not cause a significant change of the ability of learning and memory in rats.CONCLUSION1.Human and non-human-derived BDV strains have a high homology.between derived BDV strains.BDV strains had a geographical features.EBLN1 and human-derived BDV N gene has a closer genetic relationship.2.EBLN1 interference can reverse the malignant phenotype of tumor cells,suggesting that EBLN1 play the cancer-promoting role in nerve tumors.3.EBLN1 protein can increase the expression of synaptic plasticity associated proteins and active ERK signaling pathway,but its effect on learning and memory needs further studies.