| [Aims]B-cell lymphomas are solid tumors derived from B cells and are divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma.Burkitt's lymphoma(BL)is classified as a high-proliferative non-Hodgkin's lymphoma of B-cell by the World Health Organization(WHO).Related studies have found that c-Myc rearrangement of immunoglob?lin gene loci in 90%of BLs indicates that the degree of malignancy is closely related to c-Myc.In recent years,more and more transcribed ?ltra-conserved RNAs(T-UCRs)have been found to play an important role in the formation and development of tumors.Our previous study found that some of the T-UCRs in different Burkitt's lymphoma models with c-Myc expression were significantly different.This study aims to further screen for c-Myc-related T-UCRs and explore the function and mechanism of uc477+ in B lymphoma cells.[Methods]1.The 12 T-UCRs screened in the B-cell lymphoma cell line and Myc-on lymphoma tissue were detected by the strand-specific RT-PCR.After comparing with the res?lts of common RT-PCR assays,select uc477+ for further study.2.We constructed the uc.477+ overexpression plasmid uc.477+-MSCV-PIG,and then transfected the plasmid into 293T cells and packaged the virus.Infected mouse B lymphoma cell line 38B9 cells and human B lymphoma cell line Romas cell line were screened by puromycin and uc.477+ was overexpressed in cells,The positive expression rate was detected by flow cytometry,finally a B cell lymphoma cell line stably expressing uc.477+ was constructed.3.The effect of uc.477+ overexpression on the proliferation of B-cell lymphoma cells was examined by cell counting and compared with cells infected with empty vector;Flow cytometry was used to detect the effect of uc.477+ on cell cycle and apoptosis in B-cell lymphoma cells;38B9-uc.477+ and 38B9-MSCV cells were subcutaneously injected into BALB/c mice to construct a tumor model and observed the he tumor growth rate.After two weeks,the tumor tissue was removed and measured the volume and weight.4.Detection of uc.477+ host gene PLP1 expression in normal mouse B cells?mouse B lymphoma cells(38B9,myc3)and 38B9-uc.477+,38B9-MSCV cells,Explore the possible relationships between uc.477+ and its host genes,The expression of uc.12+A host gene SFPQ in B-lymphoma cell line was examined to investigate the relationship between uc.12+A and SFPQ.[Res?lts]1.Strand-specific RT-PCR res?lts showed that uc.477+,uc.12+A,uc.388+ were increased in mouse B lymphoma cells(38B9,myc3)and Spontaneous B lymphoma tissue of Eu-myc mice.The uc.477+ up-reg?lation trend was found to be significant,so we chose uc.477+ as a further study object.2.M?ltiple T-UCRs from the sense and antisense strands were selected for expression by strand-specific RT-PCR and general RT-PCR,and the res?lts were compared to verify the reliability of the strand-specific RT-PCR method.We found that uc.31+,uc.142+A,uc.468+A were down-reg?lated in the strand-specific reverse transcriptome of myc3 cell line.The expression of uc.388+ and uc.12+A in the chain-specific RT-PCR group was lower than that in the normal RT-PCR group.However,the overall trend of up-reg?lation of uc.388+ and uc.12+A in 38B9 did not change when using these two methods.This demonstrates that strand-specific RT-PCR can eliminate RNA interference from the other strand and res?lt in more accurate detection.3.After in vitro detection of 38B9 cells and Romas cells overexpressing uc.477+,compared with the control group,it was found that cell proliferation was accelerated and apoptosis was decreased.After in vivo detection of 38B9 cells overexpressing uc.477+ tumor tissue,the weight and volume were significantly increased compared with the control group..4.The res?lts of RT-PCR showed that uc.477+ host gene PLP1 was highly expressed in B lymphoma cell lines,and was increased in 38B9 cells that had overexpressed uc.477+.At the same time,it was also found that the uc.12+A host gene SFPQ increased in B lymphoma cells and was highly expressed in 38B9-uc.12+A which overexpressing uc.12+A.uc.12+A and SFPQ showed a significant positive correlation between normal B cells and B lymphoma cells.The flow assay showed that the proliferation and division of 38B9 cells after overexpression of uc.12+A were accelerated.[Conclusions]Uc.477+ promotes the growth of B lymphoma cells;uc.12+A promotes the growth of mouse B lymphoma cells. |
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