| AimsLymphoma is a malignant tumor originated from the LYMPHOPOIETIC system.It includes Hodgkin's lymphoma(HL)and non Hodgkin's lymphoma(NHL).NHL is divided into B cell,T cell and NK/T cell NHL according to the cell source.Most of NHL in clinic is B cell type.Burkitt lymphoma is a highly invasive B cell NHL derived from the germinal center of follicular cells.In this tumor,myc gene is activated due to chromosomal translocation,which promotes cell proliferation,so that normal B cells become malignant proliferating tumor cells.In the early stage of our research group,we constructed the B-cell lymphoma model induced by c-myc,and took tumor tissue as chips under the condition of c-myc activation and inactivation,respectively,to detect whether the expression of various non coding RNA changes.The expression of t-ucrs changed significantly after c-myc activation,and the expression of uc.477+changed significantly after c-myc activation.The purpose of this study is to investigate whether the ultra conserved RNA uc.477+is regulated by c-myc and to explore its mechanism in B-cell lymphoma.Methods1.Construct the B-cell lymphoma model of Myc on and myc off control mice,take the lymphoma tissues of the two groups for microarray detection,select the t-ucrs with significant difference expression,and determine uc.477+.Normal B cells were selected from the bone marrow of C57BL/6 wild-type mice.The expression of uc.477+in normal B cells and B lymphoma cells(38b9)was detected by RT-PCRNCBI looks for the 3000 bp sequence upstream of the uc.477+gene,designs primers,and performs a ChIP and dual luciferase report experiment to detect whether there is a c-Myc binding site upstream of the uc.477+gene.2.Chip technology and double Luciferase Report showed that there was a c-myc binding site at 167bp-173bp upstream of uc.477+and c-myc could activate the expression of uc.477+.3.Connect the uc.477+gene fragment to the pmsv-pig retroviral expression vector,and construct the uc.477+overexpression plasmid.477 pmsv pig and empty pmsv pig were packaged into retroviral infected mouse B lymphoma cell line 38b9 by 293T cells,and uc.477+over expression stable transformed 38b9 cell line was constructed.4.In vivo experiments:After uc.477+overexpression,the 38B9 strain and the empty vector 38B9 cell strain had a cell count of 1 × 106,respectively,and were inoculated subcutaneously in BALB/c mice.The tumor tissues were taken for comparison after 10 days.Ki-67 immunohistochemistry was used to detect the proliferation of UC.477+overexpressed 38B9 and empty vector 38B9 tumor tissue.Results1.RT-PCR showed that in 38B9 cells,the expression of uc.477+was significantly higher than that in normal B cells of mouse spleen,which was consistent with the microarray results.2.ChIP and dual luciferase report experiments detect that there is a c-Myc binding site at 167bp-173bp upstream of uc.477+and c-Myc can transcribe and activate the expression of uc.477+.3.The 38B9 cell line stably overexpressing uc.477+and the 38B9 cell line stably transfected with the MSCV empty vector were successfully constructed.4.In vitro experiment:cell count proved that overexpression of uc.477+in 38b9 cells could promote cell growth;apoptosis experiment proved that overexpression of uc.477+in 38b9 cells reduced apoptosis;cell cycle showed that overexpression of uc.477+in 38b9 cells decreased in G0-G1 cells,and increased in G2-M phase.5.In vivo tumor bearing experiment:compared with the control group,the tumor tissue weight and volume of 38 B9 cell line overexpressing uc.477+increased significantly.The results of Ki-67 immunohistochemistry showed that the over expression of uc.477+in 38b9 tumor tissue was significantly higher than that in the control group.Conclusions1.There is a c-Myc binding site upstream of uc.477+,c-Myc can transcribe and activate the expression of uc.477+.2.uc.477+has increased expression in B lymphoma cells,and has the effect of promoting the growth of B lymphoma cells in vitro and in vivo. |
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