The Role And Mechanism Of Ultraconserved RNA Uc.133 And Its Host Gene RSRC1 In B-cell Lymphomagenesis

Lymphoma,which accounts for about 3-4%of all tumors worldwide,is the seventh most common tumor and a malignant tumor of the lymphatic system.It consists of two classes of lymphoma:Hodgkin's lymphoma(HL)and non-Hodgkin's lymphoma(NHL).In China,the incidence of NHL is growing at an annual rate of 5%,while the global mortality rate for NHL is more than 50%.P53 is a tumor suppressor gene,whose mutation causes large numbers of malignancies including most of the lymphomas.Molecular targeted drugs have been confirmed to effectively improve the 5-year survival rate of patients,however,the survival rate is still low,with only about 40%.Therefore,it is of great significance to investigate the mechnism of lymphomagenesis to find novel molecular targets.Long noncoding RNAs(IncRNAs)are RNA molecules with a length of more than 200 nucleotides that cannot encode proteins,and the base composition ranges from 200bp to 100,000bp.Ultraconserved RNA(UCRs),whose sequence is 100%identical in humans,rats and mice,is one of the subcategories of LncRNAs.uc.133 is an ultraconserved RNA with a total length of 277 nucleotides,and its biological functions have not been reported so far.Rsrc1 is a gene on the third chromosome q25 and encodes an estrogen(ER)co-regulation factor consisting of a total of 334 amino acids.The Rsrcl gene contains 9 introns and uc.133 is located in the 4th introns of Rsrc1.According to the literature,RSRC1 can promote the growth of breast cancer cells MCF-7 and BT474,but its role in lymphoma has not been reported.Based on our previous studies,p53 may be able to directly regulate certain ultraconserved RNAs.The aim of this project is to elucidate:1)The relationship between ultraconserved RNA uc.133,the host gene RSRC1 and tumor suppressor p53;2)the expression patterm of ultraconserved RNA uc.133 and its host gene product RSRC1 in lymphomas;3)The function of uc.133 and RSRC1 in lymphomas;4)The possible mechanism of the role of uc.133 and RSRC1 in lymphomas.First,we examined the relationship among ultraconserved RNA uc.133,RSRC1 and tumor suppressor p53.The fresh isolated bone marrow cells from p53ER knock-in mice were mixed with the viral packaging cells E86(E86-Myc)that can secrete the oncogene gene Myc retrovirus,and then injected the mixed cell population into the mice subcutaneously.Four weeks later,palpable tumors,about 1cm3 size,developed in lice.Pathological and flow cytometric analyses showed that all tumors were of B-cell origin.In this mouse lymphoma model with the fusion of p53 gene and partial estrogen receptor gene sequence,administration of estrogen simulator tamoxifen allows p53ER fusion protein to enter the nucleus from the cytoplasm,thus activating the function of p53.In this way,we successfully established a p53-inducible mouse model for human B-lymphomas.We next grouped the mice bearing tumors,injected tamoxifen into the mice,dissected the tumors in different time periods(Omin,10min,20min,30min,60min),and extracted total RNA.Semiquantitative real-ime PCR was used to examine the RNA levels of uc.133 and RSRCI.The results showed that the expression level of uc.133 and RSRC1 in 30min changed significantly after p53 activation,suggesting that p53 may directly regulate the expression of uc.133 and RSRC1.We plan to further use anti-p53 antibodies to pull down the potential binding DNA sequences with chromatin immunoprecipitation(ChIP)assay.And also promoter luciferase report experiments will be used to verify whether p53 binds the upstream DNA sequence of the uc.133 gene or the promoter region of the RSRC1 gene.Secondly,we tested the expression of ultraconserved RNA uc.133 and its host gene RSRC1 in lymphoma cells and tissues using real-time PCR.The normal mouse B lymphocytes and murine B lymphoma cells were compared,together with 15 cases of human reactive inflammatory lymph nodes and 15 cases of human diffuse large B-cell lymphoma(DLBCL)with high Myc expression.We found that uc.133 expressed about 100 times lower in mouse lymphomas compared with normal B cells,7 times lower in human lymphoma tissues,while the expression of RSRC1,the host gene of uc.133,increased 2.5 folds in mouse lymphomas,and increased 6 folds in human lymphoma tissues.These data indicate that the expression of uc.133 and its host gene RSRC1 in B-cell tumors is significantly different from the ones in normal B lymphocytes.Thirdly,we investigated the effects of uc.133 and RSRC1 on the growth of lymphoma cells.Firstly,we constructed the retroviral plasmid of the uc.133 and the lentiviral vector of RSRC1,and the lymphoma cells were infected with either viral vectors,so that uc.133 and its host gene RSRC1 can express in mouse lymphoma cell 38B9 and human lymphoma cell Romas.The growth rates and apoptosis of the lymphoma cells were analysed after enforced expression of uc.133 and RSRC1.It was found that uc.133 significantly inhibited the growth of 38B9 and Romas lymphoma cells,while RSRC1,contrary to uc.133,promoted the growth of 38B9 and Romas lymphoma cells.After annexin V staining,it was found that the early apoptoticlymphoma cells increased after expression uc.133,while the early apoptotic lymphoma cells decreased after over-expression RSRC1.These results indicate that uc.133 and RSRC1 can affect the growth of B-cell lymphomas,but with opposite effects.Finally,we studied the possible mechanism of uc.133 and its host gene RSRC1 in lymphomas.RNA and proteins were prepared after ectopic uc.133 expression in B lymphoma cells to examine the expression level of RSRC1 by real time PCR and Western Blot.It was shown that RSRC1 was reduced about 13 folds after over-expression of uc.133.However,after enforced expression of RSRC1 in B lymphoma cells,uc.133 level was basically unchanged.This suggests that the anticancer effect of uc.133 may be carried out by inhibiting the expression of RSRC1,the host gene of uc.133,and the cancer-promoting effect of RSRC1 may be achieved by other mechanism rather than the effect of uc.133.We will continue to study the mechanism of how uc.133 and its host gene RSRC1 affect the B-lymphomagenesis,and in this way new molecular targets for therapeutics can be discovered.