Clinical Significance And Mechanisms Of Competing Endogenous RNA Networks In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia（CLL）is a highly heterogeneous,clonal proliferative malignancy characterized by the accumulation of mature B lymphocytes in peripheral blood,bone marrow,spleen and lymph nodes.In recent years,the prognosis of CLL patients has been improved with the deepening of molecular biology studies and the development of new small molecule targeted drugs,but its pathogenesis has not been fully elucidated.Drug resistance and relapse/refractory are still problems that needs to be solved urgently in CLL patients.Noncoding RNAs（ncRNAs）are products of gene transcription process,which can regulate a variety of physiological and pathological processes.Among ncRNAs,competing endogenous RNAs（ceRNAs）such as long noncoding RNAs（IncRNAs）and circular RNAs（circRNAs）,can regulate gene expression by competing with microRNA（miRNAs）.CeRNA networks play vital roles in various neoplastic diseases,however,its role in CLL remains unclear.This study performed bioinformatics and survival analyses of differentially expressed RNAs in CLL and constructed ceRNA networks to explore potential prognostic markers and provide a basis for the pathogenesis study and treatment of CLL.Part I:Analysis of RNA sequencing results and construction of risk scoring model in patients with chronic lymphocytic leukemiaObjective:The establishment of a risk scoring system helps clinicians in making clinical decision and prognostic assessment for patients.This study aims to further explore the specific RNA expression profile of CLL and establish a novel risk scoring model to provide a powerful tool for risk stratification of CLL.Methods:We included 865 de novo CLL patients to investigate RNA expression profiles.Illumina sequencing was performed on peripheral blood mononuclear cells（PBMCs）of four CLL patients,two CLL cell lines and normal B cells of six healthy donors in our center.According to univariate Cox regression,LASSO regression as well as multivariate Cox regression analyses,we established a novel risk score model in CLL patients.Immune signatures were compared between the low-and high-risk groups with CIBERSORT and ESTIMATE program.Afterwards,we analyzed the relationship between differentially expressed miRNAs（DEmiRNAs）and immunoglobulin heavy chain variable region（IGHV）mutation status,p53 mutation status and dell7p.Results:Totally,57 differentially expressed mRNAs（DEmRNAs）and 335 DEmiRNAs were identified between CLL patient specimens and normal B cells.A novel risk score model consisting of HTN3,IL3RA and NCK1 was established and validated.The concordance indexes of the model were 0.825,0.719 and 0.773 in the training,test and total sets,respectively.The high-risk group was related to del（13q 14）as well as shorter overall survival（OS）.Moreover,we identified DEmiRNAs that related to cytogenetic abnormality of CLL patients,which revealed that miR-324-3p was associated with IGHV mutation,p53 mutation and dell7p.Conclusions:In this study,bioinformatic analyses of RNA expression profiles in CLL identified differentially expressed RNAs and developed a novel risk scoring model to assess the prognosis of CLL patients.These results optimized risk stratification of CLL patients and provided potential prognostic biomarkers in CLL.Part Ⅱ:Construction of competing endogenous RNA network and validation of circRNA circ₀002078/miR-185-3p/TCF7L1axisObjective:The role of ceRNA networks in CLL still needs to be further explored.This study aimed to construct prognosis-related ceRNA networks and explore the role of circ₀002078/miR-185-3p/TCF7L1 axis in the development of CLL.Methods:The lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed based on the survival analyses and differentially expressed RNAs with targeting relationships.The function and regulatory relationship of the circRNA circ₀002078/miR-185-3p/TCF7L1 axis were verified through CCK-8 assays,cell apoptosis,cell cycle assays,and dual-luciferase reporter gene assays.Results:The survival-related lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed to further facilitate the development of potential predictive biomarkers.The expression of circ₀002078 and TCF7L1 were significantly elevated and miR-185-3p was obviously down-regulated in CLL patients.Besides,overexpression of miR-185-3p inhibited cell proliferation.Knockdown of circ₀002078 inhibited cell proliferation,promote apoptosis and G2/M phase arrest.In addition,dual-luciferase reporter gene assay proved that circ₀002078 competitively bound miR-185-3p with TCF7L1,attenuating the inhibitory effect of miR-185-3p on TCF7L1 mRNA,thereby promoting the expression of TCF7L1.Conclusions:This study provides a basis for the molecular mechanism of CLL pathogenesis and suggests new promising therapeutic targets through the construction of ceRNA network and the validation of the function and regulatory relationship of circRNA circ₀002078/miR-185-3p/TCF7L1 axis.