LIN28A Regulates The Progression And Chemotherapy Resistance Of Acute Myeloid Leukemia By CENPE

Background and ObjectiveAcute myeloid leukemia(AML)is a type of hematology malignancy that is characterized by abnormal differentiation and proliferation of hematopoietic stem/progenitor cells.The main clinical treatment strategy for AML is cytarabine(Ara-C)-based induction and consolidation therapy.However,more than half of AML patients still relapse after receiving Ara-C treatment,and a vary majority of relapsed AML patients will not respond or have a relatively weak response to subsequent standard induction chemotherapy.Therefore,it is of tremendous importance to explore the mechanism of AML occurrence,progression,and drug resistance.With uninterrupted evolution of molecular biology detection technology,researchers have discovered that the occurrence and development of AML are closely connected to abnormal gene expression.High-throughput RNA sequencing(RNA-seq)can screen out genes or pathways that are closely related to specific biological processes or disease progression.The proliferation of leukemia cells depends to a certain extent on the increase of mitotic events,in addition,genetic instability and aneuploidy caused by mitotic errors play an important role in the occurrence and development of AML.During mitosis,the key steps are proper assembly and attachment of mitotic spindles and separation of sister chromatids.The mitotic spindle checkpoint gene(SAC)plays a key role in it.As an important member of SAC,centromere-associated protein E(CENPE)moves in one direction along the microtubule trajectory and participates in the process of cell mitosis to ensure the accurate separation of chromosomes.Researches have revealed that CENPE is overexpressed in varieties of neoplasms,and interference with CENPE expression can prevent cancer cells’ growth and reverse drug-resistance.The mechanism and function of CENPE in the AML occurrence,progression,and Ara-C resistance have not been completely studied.This study is mainly divided into three parts: 1.Using RNA-seq technology to analyze the molecular biological characteristics of patients with Ara-C resistance relapsed and/or refractory AML.2.Combining RNA-seq data and TCGA/GEO database analysis data to screen out the mitosis cell cycle related gene CENPE,and further explore the impact of CENPE interference on the progression and Ara-C resistance of AML.3.Combine literature review and co-expression analysis to explore the mechanism of LIN28A/CENPE in regulating the progression and Ara-C resistance of AML.Part Ⅰ: To explore the molecular biological characteristics of patients with relapsed and/or refractory AML based on RNA-seqBackground and ObjectiveRNA-seq and bioinformatics analysis were used to analyze the gene expression profiles and biological processes of relapsed/refractory AML patients(R/R-AML),refractory secondary AML patients(S-AML),de novo AML patients(AML)and healthy controls(HC).Materials and Methods1.PBMCs were isolated from HC,patients with de novo AML,R/R-AML and S-AML.2.RNA was extracted from PBMCs,then quality,integrity and purity testing,and quantitative determination were conducted.3.After removing the r RNA,RNA was reversely transcribed into c DNA.4.The quality and content of c DNA library was measured by q PCR assay.5.Library was sequenced by Illumina Hiseq 4000.6.Low-quality data was removed after quality control.7.Data post quality control was aligned to the reference genome.Differentially expressed genes(DEGs)were screened out after analysis the expression of genes and transcripts.8.Principal component analysis(PCA)and correlation analysis of samples were performed.9.The DEGs in de novo AML versus HC,R/R-AML versus de novo AML,and S-AML versus AML were analyzed.Moreover,GO and KEGG analysis for the DEGs were conducted.Results1.Compared with HC,when fold change≥1.5 and p<0.05,there are a total of1966 DEGs(739 up-regulated and 1227 down-regulated)in de novo AML.GO and KEGG revealed that the differential genes are primarily convoluted in the pathways and biological processes of mitotic cell cycle(GO: 0000278),regelation of immune system process(GO: 0002682),cell cycle(hsa04110)and T cell receptor signaling(hsa04660).2.Compared with de novo AML,when fold change≥1.5 and p<0.05,there are a total of 188 DEGs(113 up-regulated and 75 down-regulated)in R/R-AML.GO and KEGG revealed that the differential genes are primarily convoluted in the pathways and biological processes of monounsaturated fatty acids biosynthesis(GO: 1903966),phosphatidylinositol-3-phosphate biosynthesis(GO: 0036092),regulation of immune system process(GO: 0002682),and graft versus host disease(hsa05332).3.Compared with de novo AML,when fold change≥1.5 and p<0.05,there are a total of 734 DEGs(507 up-regulated and 227 down-regulated)in S-AML.GO and KEGG revealed that the differential genes are primarily convoluted in the pathways and biological processes of cell cycle(GO: 0007049,hsa04110),mitotic cell cycle(GO: 0000278)and immune response(GO: 0006955).Conclusions1.The gene expression information of PBMCs in patients with S-AML and R/R-AML were meaningfully distinct from that of patients with de novo AML.2.DEGs in R/R-AML and S-AML patients are mainly enriched in biological processes such as mitotic cell cycle and immune system.Part Ⅱ: The effects of CENPE on AML proliferation,apoptosis,cell cycle and Ara-C resistanceBackground and Objective DEGs in recurrent AML(R-AML)and primary AML were analyzed using TCGA and GEO database.Then,CENPE was screened out in combination with the results of RNA-Seq analysis in part 1.Moreover,the effect of CEPNE interference on AML progression and Ara-C resistance was investigated in vitro.Materials and Methods 1.Gene expression data of 151 R-AML samples and 7 primary AML samples were downloaded from TCGA database and GEO database,respectively.Compared with primary AML group,DEGs(fold change≥2 and p<0.05)in R-AML were identified.2.Overlapping up-regulated genes in AML versus HC(RNA-seq analysis data from part 1),R/R-AML+S-AML versus AML(RNA-seq analysis data from part 1)and R-AML versus AML(TCGA data)were screened out by Venn analysis.3.CCK-8 assay was applied to explore the effects of CENPE,CENPF and DLGAP5 knockdown on K562 and THP-1 cell proliferation.4.Flow cytometry was used to discover CENPE interference’s function on cell cycle progression after propidium iodide(PI)staining in THP-1 cells and K562.5.CENPE interference’s influence on THP-1 and K562 cell apoptosis was analyzed through flow cytometry after PI and Annexin V-FITC double staining.6.The effect of CENPE knockdown on Ara-C resistance of K562 and THP-1 cells was measured by CCK-8 assay.Results 1.A total of 7957 DEGs with fold change≥1 and p<0.05 were identified(5964 up-regulated and 1993 down-regulated)in R-AML compared with primary AML(TCGA R-AML vs GEO AML).2.A total of 12 overlapping up-regulated genes were identified in AML versus HC(RNA-seq analysis data from part 1),R/R-AML+S-AML versus AML(RNA-seq analysis data from part 1)and R-AML versus AML(TCGA/GEO analysis data).Among these overlapping up-regulated genes,3 genes(CENPE,CENPF and DLGAP5)closely related to cell cycle progression were screened out.3.Down-regulation of CENPF led to a notable decrease in the proliferation of THP-1 cells,but had slightly effect on the proliferation of K562 cells.CENPE and DLGAP5 knockdown markedly weakened the proliferative ability of K562 and THP-1 cells.The survival activity of K562 cells after CENPE interference was significantly lower than that after DLGAP5 interference.4.In THP-1 cells and K562,down-regulation of CENPE resulted in cell cycle G1 arrest and hampered the cell cycle’s progression to G2/M phase.5.In THP-1 cells and K562,down-regulation of CENPE promoted late and early cell apoptosis.6.Down-regulation of CENPE enhanced the Ara-C sensitivity of K562 and THP-1 cells.Conclusions 1.Analysis of TCGA and GEO data shows that compared with primary AML patients,the expression of CENPE in the whole blood of R-AML patients is significantly up-regulated.2.CENPE knockdown repressed AML cell proliferation,hampered cell cycle progression,and advanced cell apoptosis.3.CENPE knockdown enhanced the sensitivity of AML cells to Ara-C in vitro.Part Ⅲ: LIN28 A regulated AML progression and Ara-C resistance through CENPEBackground and Objective Using Starbase and POSTAR databases to predict RNA-binding proteins(RBPs) that may bind to CENPE m RNA,and combining co-expression analysis and literature review to screen out RBP LIN28 A,which is closely related to tumor occurrence and development.Further explore whether LIN28 A can participate in the regulation of AML process and drug resistance by influencing CENPE expression and CENPE m RNA stability,which might provide a reliable biological target for the clinical diagnosis and treatment of AML.Materials and Methods 1.RBPs that had a possibility to interact with CENPE m RNA were predicted by Starbase and POSTAR databases.The correlation between CENPE and LIN28 A expression in AML was predicted by Starbase database.2.Gene expression data in R-AML and primary AML samples were downloaded from GEO database and TCGA database.LIN28 A expression in R-AML and primary AML samples was analyzed.3.The expressions of LIN28 A and CENPE m RNAs and proteins in K562 and THP-1 cells transfected with si-LIN28 A or si-NC were observed through western blot assays and q PCR.CENPE m RNA stability was examined by actinomycin D test.4.CENPE m RNA level enriched by LIN28 A antibody was detected by RIP assay coupled with q PCR assay.5.The interaction between LIN28 A and CENPE m RNA 3’UTR was explored via RNA pull down.6.The dual luciferase report analysis was used to explore the binding site of LIN28 A and CENPE m RNA 3’UTR.7.K562 and THP-1 cells were transfected with pc DNA3.1,pc DNA3.1-LIN28 A,pc DNA3.1-LIN28A+si-NC,or pc DNA3.1-LIN28A+si-CENPE.Cells proliferation was evaluated by CCK-8 assay.Cell apoptosis and cell cycle progression were tested by flow cytometry.After treated with varing concentrations of Ara-C,the IC50 was measured via CCK-8 assay.Results 1.Starbase and POSTAR databases analysis showed that LIN28 A had a possibility to interact with CENPE m RNA.Correlation analysis showed that LIN28 A expression was positively related with CENPE expression in AML.2.TCGA/GEO database analysis showed that compared with the primary AML group,LIN28A’s expression in R-AML samples was considerably raised.3.In comparison with si-NC-transfected cells,the CENPE and LIN28 A m RNA and protein expressions were remarkably down-regulated in si-LIN28A-transfected THP-1 cells and K562.LIN28 A interference markedly weakened the stability of CENPE m RNA in K562 and THP-1 cells.4.Compared with Ig G group,CENPE was substantially enriched by LIN28 A antibody.5.Compared with bead group or antisense strand group,LIN28 A protein was largely enriched by biotin-labeled sense strand CENPE 3’UTR.6.Compared with Wt+Vector group,LIN28 A overexpression significantly increased the luciferase activity of wild-type CENPE 3’UTR reporter.While compared with Wt+LIN28A group,luciferase activity was reduced after binding the mutated CENPE 3’UTR and LIN28 A.These data suggested that LIN28 A could interact with CENPE 3’UTR through the GGAGA binding sites.7.Compared with the pc DNA3.1 group,LIN28 A overexpression promoted cell proliferation and cell cycle progression,inhibited cell apoptosis and enhanced cell resistance to Ara-C in myeloid leukemia cells.CENPE knockdown reversed the impacts of LIN28 A on cell proliferation,cell apoptosis,cell cycle progression and Ara-C resistance.Conclusions 1.LIN28 A improved CENPE m RNA stability and increased CENPE m RNA and protein by directly binding with the GGAGA motif in CENPE 3’UTR.2.LIN28 A overexpression promoted AML progression and enhanced the drug resistance of AML cells to Ara-C by destabilizing CENPE.