The Mechanism Of Ribosomal Protein RPL34 And Its Pseudogene RPL34-PS1 Promote The Growth Of B-cell Lymphoma

Tumorigenesis is a combined process of tumor initiation,progression,invasion and metastasis,involving many endogenous and exogenous factors.It is generally acknowledged that the 'gain of function' of oncogenes and the 'loss of function' of tumor suppressor genes are the main causes of tumor formation.However,the dramatic differences in genomic geneexpression and pathological manifestations of different tumors have greatly increased thedifficulty in understanding tumorigenesis and tumor progression.B-cell lymphoma arises during different steps of B-lymphocyte development.B cell development involves severalstages and starts in the primary lymphoid organs and then differentiates in secondary lymphoid tissues such as lymph nodes,spleen or tonsils.During development,several DNA modifications that are essential for a normal immune response occur.However,these modifications may lead to abnormal gene expression and the development of B-cell lymphoma.B-cell lymphoma usually is a solid tumor derived from B cells,including Hodgkin's lymphoma and non-Hodgkin's lymphoma.There are many types of Hodgkin's lymphoma includingclassic Hodgkin's lymphoma and nodular lymphocyte predominant Hodgkin's lymphoma,which are now considered as B-cell-derived tumors Diffuse large B-cell lymphoma,follicular lymphoma,mucosa-associated lymphoid tissue lymphoma,small lymphocytic lymphoma/chronic lymphocytic leukemia,and mantle cell lymphoma are the most commonnon-Hodgkin's lymphomas,accounting for 3/4 of total non-Hodgkin's lymphoma cases.The ribosome is an indispensable cellular machine for protein synthesis and an important factory that regulates life processes.Ribosomal biogenesis is a highly complex and energy-consuming process that starts at the nucleoli.Although ribosomal proteins(RPs)play important role in ribosome assembly and protein translation,their functions independent of ribosomes have received great attention since their discovery.Scientific research has found mutations in ribosomal protein genes in different cancers.For example,mutations in the ribosomal protein gene have been found in endometrial cancer(RPL22),T-cell acute lymphocytic leukemia(RPL10,RPL5 and RPL11),chronic lymphocytic leukemia(RPS15),colorectal cancer(RPS20),and glioma(RPL5).More than a dozen ribosomal proteins have been found to activate the tumor suppressor protein p53 pathway in response to ribosomal stress.In addition,these ribosomal proteins are also widely involved in various physiological and pathological processes.However,the relationship between RP and lymphoma,especially B-cell lymphoma,is rarely reported.Pseudogene(PS)is a DNA sequence that loses its normal function due to small differences in sequence compared with its corresponding gene.It has long been considered a non-functional biological waste;butresearches have found that the RP gene can produce a large number of pseudogene transcripts,whose genesare scattered throughout the genome.Although pseudogenes are considered inactive,they have the potential to produce functionalnon-codingRNA and even proteins.At the same time,studies have shown that abnormal levelsof pseudogene transcripts can be used as predictors of some cancers.Our research group first used CD 19 magnetic beads to sort the spleen B cells fromthe miR-144/451-/-,p53 KI,and Lnk-/-triple mutant mice at the age of 2 months and remaining 7 groups of control mice,and then performed RNA sequencing andanalysis.Wefound that a pseudogene RPL34-PS1 was specifically and highly expressed in the B cells.Our group has previously shown that all the triple mutant mice die of B-cell lymphoma/leukemiawhen mice reach5months old.In order to explore the relationship of RPL34-PS1 with B cell lymphomagenesis,we have decided to construct B-lymphoma cell lines:MSCV-RPL34-PS1-38B9and pBABE-RPL34-38B9 which overexpress RPL34-PS1 RNA and RPL34 protein respectively.Then,we explored the influence of RPL34-PS1 and RPL34 on the growth of B cell lymphomasby accessingproliferationand apoptosis in vitro and measuring the size,weight,histology,mitosis and apoptosisof the xenographed tumors.Finally,the possible mechanism of tumor-promotingeffectof RPL34-PS1 is discussed in order to provide novel potential therapeutic targets for the treatment of B-cell lymphoma.Methods:1.B cells in the spleensofa total of 8 groups of mice(WT,miR-144/451-/-,Lnk-/-,P53 KI,Lnk-/-miR-144/451-/-,p53KILnk-/-,miR-144/451-/-Lnk-/-,p53 KI miR-144/451-/-Lnk-/-)were sorted based on CD19-coated magnetic beads.2.RNA-Seq analysesof spleen B cells from8 groups of mice were performed to examine abnormally up-regulated and down-regulated genes.3.The RPL34 and RPL34-PS1 sequences obtained by PCR were cloned into the retroviral vectors pBABE-Puro and MSCV-PIG,respectively,and then the plasmids pBABE-RPL34 and MSCV-RPL34-PS1 were co-transfected with the packaging plasmid pcl-eco into 293T cells to produce retroviruses.After infection ofthe mouse B lymphoma cell line 38B9 with the retrovirus,followed by selection by puromycin,the cell lines pBABE-RPL34-38B9 and MSCV-RPL34-PS1-38B9,which stably overexpress RPL34 and RPL34-PS1,were constructed and then cell proliferation,apoptosis and other functionsin vitro were investigeted.At the same time,in vivotumor bearing experiments were performed to examine the effects of RPL34-PS1 and RPL34 on the growth of B-cell lymphomasby measuring the tumor size,weight,histology,proliferation,apoptosis and other parameters.4.The expression levels of RPL34 and RPL34-PS1 in spleen B cells of 8 groups of mice,38B9,pBABE-RPL34-38B9 and MSCV-RPL34-PS1-38B9 were analyzed by chromas software,and the ratio of RPL34-PS1toRPL34was calculated.Results:1.Compared with the spleen B cells fromthe 7 control groups of mice,the spleen B cells fromtriple mutant micespecifically and highly expresspseudogene RPL34-PS12.Ectopic expression of RPL34-PS1 has no effect on the cell cycle,proliferation and apoptosis of 38B9 B-lymphoma cells in vitro.However,enforced expression of RPL34-PS1 significantly promotes the tumor growthof 38B9 in vivo.Histological analysis of tumors tissuesindicatesthat ectopic expression of RPL34-PS1 significantly increases the proliferation ability of tumor cells.Flow cytometry analysis of in vivo tumor cellsreveals a significant reduction in apoptosis.These results suggest that the function of RPL34-PS1 requires a more complicated microenvironment.3.Overexpression of RPL34 significantly increases the S-phase and proliferative capacity of the 38B9 in vitro,andalso significantly reduces the apoptotic rate of 38B9 cells.In vivotumor-bearing experiments also show that overexpression of RPL34 significantly increases the tumorigenic capacity of 38B94.The expression levels of RPL34-PS1 is higher than that of RPL34 in spleenic B cells from triple mutant mice.Even in MSCV-RPL34-PS1-38B9 cells,whose RPL34-PS1 was enforcely expressed,the levelsof RPL34-PS1 was still lower than that of RPL34,suggesting that the effect of the RPL34-PS1 in ectopic expression experiments might be underestimated,and the ratio of RPL34-PS1 toRPL34 inB lymphocytes might be an important factor for tumorigenesis of B-lymphomas.Conclusion:1.Both RPL34 and RPL34-PS1 promote the growthof B lymphomas2.RPL34-PS1 may promote tumor growth by altering the tumor microenvironment.3.Ratio of RPL34-PS1 to RPL34 expression levels might be an important factor for the development of B-lymphomas.