| B cell development is a multi-step process which coordinated by complex signal network.B cell mainly originate from embryonic hematopoietic stem cell in the embryo.In the mammal,B cell originate from pluripotent hematopoietic stem cell of bone marrow.First of all,stem cell differentiate into common lymphoid progenitor cell(CLP)after the action of cell signals.Then,common lymphoid progenitor cell develop into pro-B cell,which accompanied by immunoglobulin heavy chain(Ig H)gene rearrangement.A successfully rearrangement of Ig H chain associate with the Vpre B/5 to form the pre-B cell receptor(pre-BCR)which direct the transformtion from pro-B cell to pre-B cell.The signal of pre-BCR induce immunoglobulin light chain(Ig L)gene rearrangement.Successfully rearrangement of Ig L chain associates with Ig H to form the B cell receptor(BCR),which is the signal of immature B cell.Finally,immature B cell develop into mature B cell under the stimulation of BCR receptor signals.After encountering antigens,mature B cell undergo a series of activation steps and continue to differentiate into plasma cell or memory cell which synthesize and secrete antibodies.Abnormal development of B cell will result in immune diseases or tumor.Long noncoding RNA(lnc RNA)refer to a large class of non-protein-coding transcripts that lack of complete open reading frames.These RNAs are greater than 200 bases in length and were identified as genomic“dark matter”once.They affect the transcription and stabilization of target gene by directly binding with m RNA promoter regions.More and more evidences show that lnc RNA play an important role in the occurrence and development of many diseases.For example,lnc RNA has been reported to participate in the occurrence and development of tumor cells as a suppressor or promoter.In addition,lnc RNA is necessary to maintain the normal function of the body.For instance,lnc RNA play a critical role in the differentiation and maturation of T cells,NK cells and macrophages.In this study,we found that the expression of lnc00492 is correlated with the development and maturation of B cell.On the other hand,we confirmed that lnc00492 is a novel long non-coding RNA and that there are no reports about lnc00492 biological functions in B cell.Therefore,fully understand the function and molecular mechanism of lnc00492 in B cell development may provide a reference for the treatment of autoimmune diseases and B cell-related tumors in clinic.Research purposesExplore the biological function and molecular mechanism of lnc00492 in the development and immune response of B cells.Provide new research directions for some specifically expressed lnc RNA as a marker and therapeutic target for clinical diagnosis of immune diseases or B cell-related tumors.Methods1.Flow cytometry was preformed to isolate different stage B cells from bone marrow and spleen of mouse.Agarose gel assay was used to detect the quality of RNA extracted from B cells.2.Bioinformatic methods and q PCR technology were used to analyze and verify the expression of lnc RNA.3.Detected the basic characteristics of lnc00492 by q PCR assay,RNA FISH assay and sub-cellular fraction assay.4.Lentivirus-mediated strategy was used to knock-down the expression of lnc00492 in vitro.CCK-8 assay and CFSE assay were preformed to evaluate the function of lnc00492 in B cell proliferation,division and differentiation induced by LPS.5.CRISPR/Cas9-mediated strategy was used to build the mouse module with lnc00492deletion in vivo.Assess the effect of lnc00492 on the development,differentiation and immune response of B cells by q PCR assay and flow cytometry assay.6.Identify the directly binding protein of lnc00492 by RNA pull-down and mass spectrometry analysis method.7.Confirmed the binding sits of lnc00492 and target protein by RIP assay.Investigate the molecular mechanism of lnc00492 in B cell by western blot assay and q PCR assay.Results1.Lnc00492 is highly expressed in spleen and testis,lowly expressed in other tissues of mouse.During the development and maturation of B cell,lnc00492 is almost not expressed in Pro-B cells and highly expressed in Mature B cells and MZ B cells.The expression ratio of lnc00492 is 80%in the nucleus and 20%in cytoplasm.The coding ability predictive value of CPAT is 0.076176590835979.2.Knockdown of lnc00492 effectively reduces the expression of B cell markers include CD69,CD86 and CD138,significantly reduces the division speed of progeny B cell induced by LPS in vitro.3.Lnc00492 deficiency not impaired the proportion and numbers of Pro-B,Pre-B,Immature B cells in bone marrow and B1,B2 cells in lymph node.However,lnc00492 deficiency significantly reducted the proportion and numbers of MZ B cells.4.Lnc00492 deficiency does not affect the proportion and numbers of double negative T cells,double positive T cells,CD4~+T cells and CD8~+T cells.5.Lnc00492 deficiency significantly reduced the concentration of T cell-independent antigen-induced antibodies Ig M and Ig G3 in serum.6.Lnc00492 physically interact with co-repressor Ct BP1 in MZ B cells,which reduces its ubiquitination modification level,and reduces the degradation of Ct BP1.7.Lnc00492 deficiency significantly inhibited the expression of Hes1/5 which were downstream molecules of Notch-2 signaling pathway and hinder the transduction of Notch-2signaling.ConclusionLnc00492 is a newly discovered long non-coding RNA which mainly located in the nucleus of B cell.Its expression is positively correlated with the development and maturation of B cells.Knockout of lnc00492 resulted in the blocked development of transitional B cells to marginal zone(MZ)B cells in the spleen and impaired T cell-independent immune response.Preliminary mechanistic studies have shown that lnc00492 plays an important role in the development and maturation of B lymphocytes in the mouse spleen,promoting the ubiquitination of the co-repressor Ct BP1,which in turn regulates the Notch2 signaling pathway. |
