| AimsB-cell lymphoma is a type of tumor that occurs in lymph nodes and other lymphoid tissues and is divided into two categories,Hodgkin's lymphoma(HL)and non-Hodgkin's lymphoma(NHL).The main subject of this study is non-Hodgkin's lymphoma.The c-Myc gene is a proto-oncogene that promotes DNA repair and regulates the cell cycle,thereby affecting cell proliferation,differentiation,and apoptosis.In recent years,more and more studies have shown that the c-Myc gene plays an important role in the occurrence,development and treatment of non-Hodgkin's lymphoma.The research team constructed a B-cell lymphoma model induced by c-Myc in the early stage,and detected the differential expression of the transcribed ultraconserved regions(T-UCRs)when c-Myc activated and inactivated by chip technology.It was found that there were significant differences in the expression levels of several T-UCRs,one of which was uc.12+A.This study aims to study the effects of uc.12+A on the growth of B-cell lymphoma.Then,we will further explore its mechanism and effect.Methods1.First,we built two mouse tumor models,One group was Myc-on,and the other group was Myc-off.Then we use the chip to detect expression levels of T-UCRs.Finally,we selected one of the differentially expressed T-UCRs uc.12+A to do further research.The expression levels of uc.12+A in B lymphoma cell lines were detected by common RT-PCR and strand-specific RT-PCR,and then we observed whether the results were consistent with the results of chip.2.Construction of retroviral plasmid:According to UCbase website,we obtained the sequence of the uc.12+A,the gene sequence was amplified by PCR,then we inserted the gene sequence into the pMSCV-PIG vector by double endonuclease digestion.Thus,a recombinant plasmid containing the gene of interest is obtained.3.Cell transfection and infection:The constructed vector uc.12+A-pMSCV-PIG was transfected into 293T cells by PEI transfection,and the virus-containing supernatant was collected for 36 h,48 h,60 h,72 h.The collected virus solution is used for cell infection.The virus-containing supernatant was used to infect 38B9 cells and Romas cells,respectively,and the infection efficiency was enhanced by polybrene.After 48 hours,appropriate cells were taken and the infection efficiency was measured by flow cytometry,followed by screening with puromicin until the infection efficiency reached 80%.4.In vitro and in vivo experiments:The infected cells were divided into two groups,the MSCV control group and uc.12+A overexpression group.The effect of over-expressing uc.12+A on the proliferation of B lymphoma cells was detected by cell counting method.The total number of cells was counted for four days and analyzed by statistics;using flow cytometry to detect the effect of overexpression of uc.12+A on cell cycle and apoptosis of B cell lymphoma;cells overexpressing uc.12+A and MSCV were injected subcutaneously into mice,12 days later,the tumors were taken and compared in size and weight to evaluate the effect of overexpression of uc.12+A on the growth of B cell lymphoma.5.Find the host gene of uc.12+A through the biological website,and determine that the host gene of uc.12+A is SFPQ(splicing factor proline and glutamine rich).RT-PCR was used to detect the mRNA expression level of SFPQ in 38B9 and Romas cells overexpressing uc.12+A,and compared with MSCV control group;western-blot was used to detect the protein expression level of SFPQ in 38B9 and Romas cells overexpressing uc.12+A,which was also compared with the MSCV control group.Results1.RT-PCR results showed that the expression of uc.12+A was significantly higher in 38B9 and myc3 cells than in mouse bone marrow B cells,which was consistent with the chip.Using common PCR and strand-specific PCR to detect the expression of uc.12+A,comparing the results of two groups showed that the overall trend was consistent,that is,the expression of uc.12+A increased in lymphoma cells,but the expression of the strand-specific PCR group was significantly lower than that of the normal RT-PCR group.The results of strand-specific PCR detection of uc.12+A expression were more accurate,which could exclude the interference of the sense strand on the detection results.2.The results of in vitro cell counting showed that the proliferation rate of cells overexpressing uc.12+A was higher than that of MSCV control group,indicating that the proliferation rate of B cell lymphoma was accelerated after overexpression of uc.12+A.Cell cycle experiments showed that after overexpression of uc.12+A,the proportion of cells in S phase increased,suggesting that cell proliferation and division were vigorous;Apoptosis experiments showed that the rate of apoptosis decreased after overexpression of uc.12+A;Tumor-bearing experiments in mice showed that cells overexpressing uc.12+A grew faster in mice.All of the above experiments indicated that overexpression of uc.12+A promoted the growth of B cell lymphoma.3.RT-PCR results showed that the expression level of SFPQ was also increased in the case of overexpression of uc.12+A,and they were positively correlated.Western-blot detection of overexpression of uc.12+A group and MSCV control group showed that the expression level of SFPQ protein was increased when overexpressing uc.12+A,the trend consistent with RT-PCR results.Conclusion1.uc.12+A is overexpressed in B cell lymphoma,and it can promote the growth of B lymphoma cells in vivo and in vitro.2.As a host gene of uc.12+A,SFPQ has a positive correlation with the expression of uc.12+A. |
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