Clinical Implications And Molecular Mechanisms Of Biological Behaviors Related Biomarkers EZH2 And SPRR1A In Leukemia And Lymphoma

Introduction: Acute myeloid leukemia(AML)and diffuse large B cell lymphoma(DLBCL)are common malignant proliferative diseases in Hematology,which seriously threaten people's health.Although the current therapy has improved the clinical efficacy and prognosis of the patients,but still cannot effectively reduce the recurrence and progress of this two diseases.In recent years,with the development of molecular biology and the further research on the mechanism of disease occurrence,development,prognosis and drug resistance,many new therapeutic targets have emerged,which provides new experimental basis for the accurate treatment.In lymphoma diagnosis,the emergence of these new prognostic indicators also provides an important reference for the treatment of lymphoma.Based on the clinical practical problems and the clinical treatment plan and case information,the key molecules in the diagnosis and treatment of disease will be found,which will provide experimental basis for the development of new therapeutic methods.In January 2015,the president of the United States of America,Barack Hussein Obama proposed "Precision Medicine Plan" in the State of the Union,it is hoped that by analyzing the genetic information of about more than 1 million American volunteers,diseases can be understood from genome,the causes and therapeutic targets of diseases are found out,which paved the way for the development of targeted drugs and the achievement of precision employing pesticide to cure cancer and other diseases.Genes are DNA or RNA fragments that carry genetic information,which can synthesize protein through transcription-translation process,or control the performance of individual organisms by affecting the synthesis and biological functions of proteins;the interpretation of genes is to decipher all cipher codes of human beings.The incidence,development,prognosis and drug resistance of common human diseases,such as malignant tumors and neurodegenerative diseases,can be attributed to the abnormal expression of genes.Therefore,how to accurately identify and regulate the expression levels of abnormal genes is one of the keys to the treatment of disease.In recent years,with the continuous emergence and extensive clinical application of targeted therapies for different tumors,the therapeutic effect of malignant tumors had been greatly improved.Targeted therapeutic drugs plays a very important role in precision medical treatment by regulating the expression levels of target genes to achieve the purpose of treating disease.However,the discovery of target gene often depends on the experimental experience of researchers,through the screening of target genes and biological experiments to verify the authenticity of guesswork.Such empiricism leads to low hit rate of screening genes,which not only wastes time,manpower and material resources,but also leads to the missing of key genes.The reason is that every gene is often not isolated,and one gene could affect the expression of other genes,and is influenced by other genes.The regulatory relationships of interaction and mutual restriction in genome,which constitutes a whole genome regulatory network for nonlinear regulation relationship containing tens of thousands of genes,tens of millions of genes,and the complexity is far beyond brain's reasoning ability.Therefore,by analyzing the whole genome regulatory network,we could find information about gene-gene interaction,gene regulatory network upstream and downstream,multi-gene common signaling pathway and key genes of signal bypass,which provides information for disease pathogenesis and treatment strategies.We will talk about our previous research work from the above two aspects in this paper.Materials and methods: 1.Cell lines and patient selection: The AML cell lines used in this study included THP-1(acute monocyte leukemia),and HL60 and Kasumi-6(acute myeloid leukemia).The present study included patients with a pathologically confirmed DLBCL diagnosis who were treated in the First Hospital of China Medical University,Fourth Hospital of China Medical University,and Liaoning Province Cancer Hospital between January 1,2001,and December 31,2007.Disease dissemination was evaluated before treatment by physical examination,bone marrow(BM)biopsy,and computed tomography of the chest and abdomen.Patients were staged according to the Ann Arbor system.The number of extranodal sites and larger tumor mass diameters were also determined.Methods: Apoptosis was measured using annexin V-FITC and propidium iodide(PI)double staining,and the percentages of spontaneously apoptotic cells were determined by flow cytometry.Purified AML cells were transfected with human EZH2-specific or control si RNAs using Lipofectamine 2000 Reagent(Invitrogen,Carlsbad,CA,USA) according to manufacturer instructions.The EZH2-silenced(EZH2-si)and si RNA-transfected control(EZH2-c)cells were harvested at 48 h post-transfection,and the efficacy of EZH2 silencing was determined by western blot.The EZH2-sh and EZH2-c AML cells were collected at 48 h post-transfection,and their total proteins extracted using a total protein extraction kit(Pro Mab,Richmond,CA,USA),followed by centrifugation.The relative levels of targeted proteins to the control(GAPDH)were determined using Immu Ne software.To identify immunopositive staining for SPRR1 A,SPRR1A expression was first classified semiquantitatively according to the following criteria: 0,<1% of cells discretely expressed SPRR1A;1+,?1 and <10% of cells discretely expressed SPRR1A;and,2+,?10% of cells discretely expressed SPRR1 A.Samples scored as 1+ or 2+ were considered positive.Statistical analyses: Data from all eligible patients were analyzed for OS.Survival curves were estimated by the Kaplan–Meier method and treatment comparisons were made by the log-rank test.We analyzed the OS and compared the differences between groups using stratified log-rank tests.Hazard ratios were determined using the stratified Cox proportional-hazards model in order to assess risk factors.Two-sided p values were calculated for all tests and a p < 0.05 was considered statistically significant.Statistical analyses were performed using Graph Pad Prism 5 and SPSS software,version 19.0.Gene analysis methods should include three steps that gene selection,gene regulatory networks modeling and gene regulatory networks analysis.In gene selection we chose the mutual information method and proposed a more simplified computation.In this step,we select several genes as the relevant genes.And then,based on these relevant genes,the gene regulatory networks were established using a Dynamic Bayesian Network Model.Third,we analyzed the centrality of gene regulatory networks.Finally,we also analysed key genes in AML and DLBCL by the steps above.Results: For AML: we compared EZH2 protein levels before and after treatment with 10 ?M ATO for 72 h of THP-1,HL60,and Kasumi-6 cells.Our analysis indicated that EZH2 was significantly upregulated following ATO treatment.The percentages of apoptotic cells following ATO treatment were significantly higher relative to those without ATO treatment.Following treatment of EZH2-sh THP-1,HL60,and Kasumi-6 cells with 2.5 ?M ATO,we observed increased levels of apoptosis,suggesting that EZH2 knockdown sensitized AML cells to ATO-induced apoptosis.The relative ratios of phosphorylated to unphosphorylated GSK-3b in EZH2-sh THP-1,HL60,and Kasumi-6 cells were significantly lower relative to those observed in the EZH2-c cells,suggesting that EZH2 may promote GSK-3b phosphorylation in these cell lines to enhance Wnt/b-catenin activation.For DLBCL: the median age of the population was 56 years.Except for histologic subtype,the clinical characteristics were well balanced between the 2 groups(SPRR1A-and SPRR1A+ groups).We found that the lymphoma tissue was positive for anti-SPRR1 A staining in 305(31.54%)cases.The 5-year survival rate was significantly lower in the SPRR1A+ group(26.9% [95% CI,21.6–32.2%])than in the SPRR1A-group(53.2% [95% CI,48.9–57.5%];P < 0.001).The hazard ratio for death was 1.792(95% CI,1.364–3.778;P < 0.001)in the SPRR1A+ group.After adjustment for the 11 baseline variables with the use of Cox regression analysis,the hazard ratio remained similar.Expectedly,the multivariate analyses showed that clinical stage,lactate dehydrogenase level,and SPRR1 A expression were independent prognostic factors.Gene analysis methods included three steps that gene selection,gene regulatory networks modeling and gene regulatory networks analysis.In gene selection we chose the mutual information method and proposed a more simplified computation.In this step,we select 40 genes as the relevant genes.And then,based on these relevant genes,the gene regulatory networks were established using a Dynamic Bayesian Network Model.Third,we analyzed the centrality of gene regulatory networks.Finally,we also analysed key genes in AML and DLBCL by the steps above.Conclusion: Our results suggested that EZH2 in leukemic cell lines might inhibit ATO-induced apoptosis,and that EZH2 may be a potential therapeutic target in AML patients undergoing ATO treatment.Our findings provide new insights into the role of ATO and EZH2 in regulating AML progression.SPRR1 A expression may be useful as a prognostic factor for diffuse large B-cell lymphoma.Gene analysis methods including three steps that gene selection,gene regulatory networks modeling and gene regulatory networks analysis are effective methods for gene expression regulation analysis.