The Selection And Function Study Of LncRNAs Related To The Development Of B Cells

Objective:To screen long non-coding RNAs related to B cell development in immature and mature B cells obtained by magnetic cell sorting,and to investigate the effects and mechanisms of these long non-coding RNAs during B lymphocytes development.Methods:We first seperate the spleen,femur and tibia of mice,then we obtain spleen and bone marrow lymphocytes by grinding the spleen and rinsing the bone marrow respectively.B cell specific markers B220 and CD 19 were amplified by ordinary PCR to identify the presence of B cells in both lymphocytes.IgM+ IgD" immature B cells and IgM+ IgD+ mature B cells were received from spleen and bone marrow lymphocytes by magnetic cell sorting respectively,according to the expression of B cell surface receptors.Cell immunofluorescence and flow cytometry were used to identify the correct cell sorting.The relative expression of those candidate LncRNAs,namely,GAS5、TUG1、MALAT1、Linc-P21 and HOTAIR,which associated with B-cell lymphoma,as well as other nine non-coding RNAs specifically expressed in B cells both was detected by ordinary PCR and RT-qPCR in two B lymphocytes.SiRNA was designed and transfected into two kinds of B cells after screening for long non-coding RNA with significant differences in expression levels,and RT-qPCR was used to amplify the B-cell specific surface molecμles among immature and mature B cells.We also apply cell flow cytometry to investigate the effect of different SiRNAs on B cells development after transfection.Resμlts:(1)B220 and.CD 19 can be amplified in these two lymphocytes,it means we sort the lymphocytes in a corret way.FITC-IgM and PE-IgD added in both lymphocytes were detected by flow cytometry.The ratio of immature B cells in spleen lymphocytes was found to be about 15%and that of mature B cells was 35%;immature B cells in bone marrow lymphocytes accounted for 30%,while the proportion of mature B cells was only 12%.(2)The immature and mature cells obtained by magnetic cell sorting were identified to be corret by cell immunofluorescence.The purity of immature B cells after flow analysis change from 30%to 80%,and that of mature B are also from 35%to 82%.(3)RT-qPCR was used to detect the relative expression levels of several candidate long non-coding RNAs in immature and mature B cells.We find that HOTAIR can’t express in both B cells.GAS5,TUG1 and MALAT1 are up-regμlated by 14-fold,7.5-fold and 4-fold during B-cell maturation,respectively.While the expression of Linc-P21 was down-regμlate about 7-fold.The other nine non-coding RNAs specifically express in B cells,we also find several LncRNAs with significant differences in expression levels.(4)The SiRNAs of GAS5 and TUG1 are designed and transfected into two B cells.We find that transfection is better in mature B cells.The expression level of B cell-specific surface molecμles is detected by RT-qPCR,but no significant difference is found.The ratio of mature B cells decresed after GAS5 SiRNA interference,while the number increased after TUG1 SiRNA interference by flow cytometry.But there is no significant effect on immature B cells while different SiRNAs interference.Conclusions:(1)The number of mature B cells in spleen lymphocytes is dominant,while immature B cells are mainly present in bone marrow lymphocytes.We find that IgM+IgD+ and IgM+IgD-can obtain mature B cells and immature B cells respectively.(2)Long non-coding RNA GAS5,TUGland MALAT1 are significantly up-regμlated during B cells maturation,while Linc-P21 is significantly down-regμlated.(3)There is no significant effect on the development of B cells from the expression of B cell specific surface molecules after transfection of GAS5 and TUG1-SiRNAs in both B cells.The ratio of mature B cells decresed after GAS5 SiRNA interference.But there is no significant effect on immature B cells while SiRNAs interference.