The Effect And Mechanism Of Ultraconserved RNA Uc.106 And Methylrosinate In B Cell Lymphoma

Lymphoma is a malignant tumor derived from the lymphoid hematopoietic system,ranking seventh among common tumors and accounting for 3-4%of all tumors.It consists of two broad categories:Hodgkin's lymphoma(HL)characterized by Reed-Sternberg cells(RS cells);non-Hodgkin's lymphoma(NHL)characterized by B cells and T-cell lymphocytes.The 5-year survival rate for Hodgkin's lymphoma in the United States is 85%,and non-Hodgkin's lymphoma is 69%.In China,the number of patients with non-Hodgkin's lymphoma increases at a rate of 5%per year.The number of incidence rate now has risen to 6.43/100,000 from 2/100,000 in the last century.There were 566 000 new cases worldwide in 2012,with 305,000 deaths.It is imperative to develop an effective treatment plan according to different subtypes of lymphoma.With the continuous study of the biological characteristics of lymphoma,the application of molecularly targeted drugs has effectively improved the 5-year survival rate of patients.Therefore,it has a crucial significance to find new tumor markers and study the mechanism of lymphoma.Long non-coding RNA(IncRNA)is a type of RNA that is more than 200 nucleotides and cannot encode proteins,whose base pair ranges from 200 bp to 100,000 bp.Many studies have found that they are highly tissue-specific and can participate in various physiological and pathological processes through transcriptional regulation,mRNA processing,sponge action,and nuclear transport.In the process of tumor development,they participate in the proliferation,invasion and metastasis of tumor cells as tumor genes or tumor suppressor genes.The specific expression of LncRNAs in tumors and their special biological functions in tumors suggest that they can be used as molecular markers and new therapeutic targets for clinical diagnosis and treatment.The ultraconserved RNAs(T-UCRs)are a type of IncRNA with base lengths ranging from 200 bp to 799 bp.All the UCRs are 100%identical among human,rat and mouse.The ultraconserved RNA uc.106,uc.102,uc.103,uc.104 and uc.105 are located in the intron of the coding gene OLAl and uc.102 overlapes with the partial sequence of the first exon.It reported in the literature that in patients with prostate cancer,the expression level of uc.106 is correlated with Gleason score and metastasis of prostate cancer.Furthermore,the genes related to cell proliferation,movement and immunity are altered after knocking down uc.106.These indicates that uc.106 plays a role in the development of prostate cancer.OLA1 is a member of the Obg family and the P-loop GTPase YchF subfamily.The OLAl/YchF protein is highly conserved during the evolution from bacteria to humans.It has been reported that OLA1 can regulate cellular oxidative stress and heat shock response,but the relationship between OLA1 and tumor is not clear.Methylrosmarinate(MR)is a kind of natural active ingredient isolated from the Labiatae plants,such as Perilla frutescens,Cassia tomentosa and so on.Recent studies have found that methylrosmarinate has a strong antioxidant stress effect,but the effect and molecular mechanism of methylrosmarinate in tumor remains unclear.In this study,the expression of uc.106 in paraffn tissue of diffuse large B-cell lymphoma and B-cell lymphoma cell lines was detected.And the effects of the uc.106 on the development of B cell lymphoma were studied through experiments in vivo and in vitro.We used the simulated human Burkitt'S lymphoma model to detect the expression level of uc.106 by activing the p53,and discovered the molecular mechanism that how the uc.106 surpress the tumor development.Finally,we studied the intervention effect of methylrosmarinate on lymphoma,providing a new target and theoretical foundation for the treatment of lymphoma.Part I.The expression level of uc.106 in diffuse large B-cell lymphoma and mouse B-cell lymphoma cell linesObjective:To detect the expression level of uc.102-106 in diffuse large B-cell lymphoma paraffin tissues and mouse B-cell lymphoma cells.Methods:(1)Collected 20 paraffin-embedded tissues of patients with diffuse large B-cell lymphoma and 20 normal lymph node tissues as control from the department of pathology,Jiangdu First People's Hospital,Yangzhou City;(2)Extracted the total RNA from bone marrow B cells in wild-type BALB/c mice and mouse lymphoma cells(38B9 cell and MYC3 cell);(3)The allele-specific qRT-PCR assay was used to detect the expression of uc.102-106 in diffuse large B-cell lymphoma paraffin tissues and mouse B-cell lymphoma cells.Results:(1)Compared with bone marrow B cells of wild-type BALB/c mice,uc.102,uc104 and uc.106 were down-regulated in mouse lymphoma cell lines 38B9 and myc3,especially the uc.106 was most significantly.However,the expression of uc.103 and uc.105 was increased;(2)Compared with the normal lymph node tissues of the control group,uc.106 showed a decreased expression in the diffuse large B-cell lymphoma paraffin tissues.Conclusion:uc.106 may exist as a tumor suppressor gene in B cell lymphoma.Part ?.The effect and mechanism of uc.106 in B cell lymphoma.Objective:To investigate the effect of uc.106 on the proliferation of B cell lymphoma cell line 38B9 and Romas and to explore its mechanism.Methods:(1)uc.106 sequences was cloned into the MSCV-pig vector;(2)uc.106 was overexpressed by infecting B cell lymphoma cell line 38B9 and Romas with retrovirus;(3)Cell count,CCK-8 cell proliferation assay and flow cytometry for GFP assay were used to detect the effects of uc.106 on the proliferation of B cell lymphoma cell line 38B9 and Romas;(4)Flow cytometry assay was used to examine the function of uc.106 on the cell cycle of B cell lymphoma cell line 38B9 and Romas;(5)Subcutaneous tumor-bearing experiment was used to verify the effect of uc.106 on the tumor growth rate and size in mice;(6)The allele-specific qRT-PCR assay was used to detect the differences of the expression level of uc.106 before and after p53 activation;(7)qRT-PCR assay was used to detect the differences of OLA1 mRNA levels before and after p53 activation;(8)Chromatin immunoprecipitation assay(CHIP)was used to validate the relationship between p53 and uc.106 promoter regions;(9)Dual-Luciferase Reporter assay was used to verify the association between p53 and uc.106 promoter regions;(10)Westemblot assay was used to examine the effect of uc.106 on OLA1 protein level.Results:(1)The expression of uc.106 in lymphoma cells 38B9 and Romas increased nearly 900 times after infected with retrovirus;(2)It's confirmed that uc.106 inhibited lymphoma cell line 38B9 and Romas proliferation by cell count,CCK-8 and flow cytometry for GFP assays;(3)Cell cycle experiments showed that the number of cells in G0/G1 phase increased after overexpression of uc.106;(4)Subcutaneous tumor-bearing experiments in mice confirmed that mice overexpressing uc.106 developed tumors later,and the tumors were lighter in the weight;(5)p53 was activatedby intraperitoneal injection of tamoxifen in the p53ER3 mouse lymphoma model,and the expression of uc.106 was measured at different time points(0 min,10 min,20 min,30 min,and 60 min).The expression level of OLA1 did not change at different time points,except for a slightly decrease in 20 min;(6)CHIP experiments indicated that p53 binds to the promoter region that is the 2000 bp upstream of uc.106;(7)Dual-Luciferase Reporter assay confirmed that p5 3 has a direct binding relationship with the promoter region that is the 2000 bp upstream of uc.106;(8)The protein expression level of OLA1 was increased after overexpression of uc.106 in B cell lymphoma cell line 38B9 and Romas.Conclusion:uc.106 inhibits B cell lymphoma cell proliferation both in vitro and in vivo.One of the mechanisms may be that activation of p53 promotes the transcription of uc.106,following the overexpression of uc.106 increased the protein level of OLA 1 which can form the OLA1/BRCA1/BARD1/p53 complex to promote cell apoptosis.Part ?.The effect and mechanism of methylrosmarinate in B cell lymphomaObjective:To study the effect of methylrosmarinate on the proliferation of B cell lymphoma cell lines 38B9 and Romas as well as a preliminary discussion of its mechanism.Methods:(1)CCK-8 cytotoxicity assay was used to detect the IC50 of methylrosmarinate in B cell lymphoma cell lines 38B9 and Romas;(2)CCK-8 cell proliferation assay were used to detect the effect of methylrosmarinate on the proliferation of B cell lymphoma cell lines 38B9 and Romas;(3)Detection the expression of uc.106 in 38B9 and Romas cells which treated with different concentration of methylrosmarinate(0,40,80 and 160 ?mol·L-1)for 24 hours by quantitative PCR.(4)Flow cytometry was performed to detect the effect of methylrosmarinate on the apoptosis and cell cycle of B cell lymphoma cell lines 38B9 and Romas;(5)Western blot assay was used to detect the effect of methylrosmarinate on the expression levels of autophagy marker proteins Bclin-1 and LC3 in B cell lymphoma cell lines 38B9 and Romas.Results:(1)The IC50 of methylrosmarinate for 38B9 cells and Romas cells on 24 h were 110.32 ?mol·L-1 and 116.32 ?.mol·L-1 respectively;(2)Methylrosmarinate inhibited the proliferation of 38B9 cells and Romas cells in a concentration-dependent manner;(3)The expression of uc.106 in 38B9 cells and Romas cells treated with different concentrations of methyl rosmarinate(0,40,80 and 160 micromol.L-1)did not change significantly after 24 hours;(4)Methylrosmarinate can promote the early apoptosis of 38B9 cells and Romas cells in a concentration-dependent manner;The cell cycle assay shows that the cells in G1 phase increased significantly after treatment with methylrosmarinate;(5)After treatment with different concentrations of methylrosmarinate,the protein levels of Beclin-1 and LC3 ? increased in a concentration-dependent manner and the ratio of LC3 ?/LC I increase significantly in the 38B9 cells and Romas cells.Conclusion:Methylrosmarinate significantly inhibited the proliferation of 38B9 and Romas cells,also simultaneously promoted apoptosis.The effects of methylrosmarinate may be due to the autophagy by increasing the expression of Beclin-1 and LC3 ? proteins.