The Roles Of AF1q,CD44 And CircRNA In Leukemia And Related Mechanisms

Section ?The study of AF1q regulating CD44 in cell proliferation and drug resistance of CMLBackground:Chronic myeloid leukemia?CML?is a hematopoietic stem/progenitor cell disorder characterized by the t?9;22??q34;q11?translocation and the formation of the fusion oncogene BCR-ABL.The BCR-ABL oncoprotein has constitutive tyrosine kinase activity that directs HSC differentiation toward myeloid progenitors and differentiated myeloid cells expansion,and is essential for the growth of CML cells.Tyrosine kinase inhibitors?TKIs?could specially target the BCR-ABL oncoprotein and largely improve the prognosis of CML patients.However,TKI therapy is not curative,as drug resistance and disease relapse are becoming a rising clinical issue.Therefore,it is necessary to identify additional therapeutic targets in CML to overcome TKI resistance as well as relapse.The AF1q/MLLT11 gene,located at chromosome 1q21,is identified as a mixed-linage leukemia?MLL?fusion partner.AF1q is found over-expressed in both acute myeloid leukemia?AML?and acute lymphoblastic leukemia?ALL?patients and acts as a poor prognostic marker for AML and myelodysplastic syndrome?MDS?,which indicates that AF1q may play an important role in leukemia.What's more,our previous study showed an elevated expression of AFlq in CML,particularly high in CD34+ CML stem/progenitor cells.However,the function and mechanism of AFlq in CML remains unclear.Objective:The aim of this study is to explore the expression profile of AF1q in different phases of CML patients and identify the effect of AFlq overexpression on cell proliferation and drug resistance in CML cells.Furthermore,we also intend to clarify the potential mechanism of AF1q in CML,thus providing the basis for a new molecular targeted therapy.Materials and methods:1.Sample collection and cell isolation:Bone marrow samples from 149 CML patients and 15 control donors were obtained,from which mononuclear cells were separated.13 CML BM CD34+ cells and 7 normal BM CD34+ cells were isolated by using a human CD34 Microbead Kit.2.Detection of AF1q expression:Real-Time RT-PCR was performed to detect the expression level of AF1q mRNA in CML primary cells and CD34+ cells from different phases of CML patients and control donors.3.Construction of AF1q expression plasmid and synthesis of siRNA sequence:The lentivirus particles were produced by cotransfecting 293T cells with lentivirus vector pLOC-AF1q or pLOC-NC;AF1q specific siRNAs and scrambled control sequences were synthesized.4.Transfection of CML cell lines,primary cells and CD34+ cells:K562 cells infected with lentiviruses harboring AF1q?K562-AF1q?or negative control?K562-NC?were selected by blasticidin for at least 2 weeks;AF1q specific siRNAs?AF1q siRNA?and scrambled control?scramble?sequences were transfected into K562/G01 cells,CML primary cells and CD34+ cells using Lipofectamine 2000 reagent.Real-Time RT-PCR and western blot were performed to verify the efficiency of transfection.5.Cell proliferation,apoptosis and viability assays:Transfected cells were seeded into culture plates and CCK8 assay was used to examine the proliferation;48h after adding IM,CCK8 assay and flow cytometry were performed to examine cell viability and apoptosis,respectively.6.Mice experiment:K562-AF1q and K562-NC cells were injected via tail vein into irradiated?1.5 Gy?NOD/SCID immunodeficient mice.4 or 8 weeks after,marrow contents of femurs and spleen cells were checked for human K562 cell engraftment by staining with anti-human CD33 antibody using flow cytometry.7.Microarray analysis:Gene expression profiles were detected to identify differentially expressed mRNAs between K562-AF1q and K562-NC cells,followed by the GO and pathway analysis.8.Detection of CD44 expression:The differentially expressed CD44 from the microarray result was selected as a candidate molecule regulated by AFlq.Real-Time RT-PCR was performed to detect the expression level of CD44 mRNA in CML primary cells and CD34+ cells from newly-diagnosed CML CP patients.The correlation was analyzed between AF1q and CD44.9.Blockage of CD44 activity:A specific CD44 monoclonal antibody was added to K562-AF1q cells to block CD44 activity,followed by cell proliferation,apoptosis and viability assays.Results:1.Aberrant expression profile of AF1q in CML patients:a markedly upregulated level of AF1q was found in any stage of CML patients,distinguishable from the controls,and AP1q expression was significantly higher in chronic phase?CP?,accelerated phase?AP?and blast phase?BP?patients compared to CCyR patients.Interestingly,for patients who achieved CCyR,AF1q expression was still higher,significantly different from the controls.We further found that the level of AFlq was elevated in CML CD34+ cells compared with normal CD34+ cells and CML CD34-cells,indicating AF1q may play an important role in CML leukemia stem cells?LSCs?.2.Effects of AF1q knockdown on IM sensitivity and IM-induced apoptosis:After down-regulation of AF1q in CML primary cells and CD34+ cells,CCK8 showed that IM sensitivity of the cells significantly increased;meanwhile,flow cytometry showed an enhanced cell apoptosis induced by IM.3.Effects of AFlq overexpression on cell proliferation,IM sensitivity and IM-induced apoptosis:After up-regulation of AF1q in the IM-sensitive cell line K562,CCK8 showed that the cell proliferation was accelerated,and IM sensitivity of the cells significantly decreased;meanwhile,flow cytometry showed a reduced cell apoptosis induced by IM.For comparison,we also examined the effects of AFlq in a K562-homologous but IM-resistant cell 'line,K562/G01.After down-regulation of AF1q in this cell line,CCK8 showed that the cell proliferation was decelerated,and IM sensitivity of the cells significantly increased;meanwhile,flow cytometry showed an enhanced cell apoptosis induced by IM.These results demonstrated that AF1q is involved in the regulation of CML cell proliferation,drug sensitivity and apoptosis.4.Overexpression of AF1q enhances engraftment of CML cells in vivo:Generally,less K562 cells were found in the BM compared to the spleen.Mice in the K562-AF1q group exhibited more K562 cells in spleen than those in the K562-NC group after both 4 weeks and 8 weeks,while there was significance in BM only after 8 weeks.5.Microarray results:By performing the genome-wide microarray assay,we found 582 genes differentially expressed after AF1q overexpression,including 465 upregulated and 117 downregulated genes.GO and KEGG pathway analysis revealed roles of AFlq in transcription regulation,signal transduction,adhesion,apoptosis,and key pathways like Ras,PI3K-AKT,mTOR pathway.Among the differentially expressed genes,CD44 was chosen as the downstream candidate of AFlq since its significant expression and close relationship with leukemia pathogenesis and drug resistance.6.CD44 expression and its correlation with AF1q:In the BM primary cells and CD34+ cells from newly-diagnosed CML CP patients,a significant positive correlation was observed between AF1q and CD44 expression.Besides,Inhibition or overexpression of AF1q in CML cells could cause a changed CD44 expression,correspondingly.What's more,we found CD44 was also significantly elevated in CML CD34+ cells compared to CD34-cells.These results indicated that AF1q could positively regulate the expression of CD44.7.AF1q functions in CML by regulating CD44:Blocking of CD44 in the K562-AF1q group resulted in significantly slowed proliferation,which was accelerated by AF1q overexpression.CCK8 also showed a modest but significant increase in IM sensitivity;meanwhile,flow cytometry demonstrated an enhanced cell apoptosis induced by IM.Conclusion:1.A higher level of AF1q expression was found in any stage of CML patients,distinguishable from the controls,indicating that AF1q could play important roles in the pathogenesis and development of CML.2.Disregulation of AF1q expression could alter cell proliferation,IM sensitivity and IM induced apoptosis,which verified that overexpressed AF1q in CML could promote cell growth,induce IM resistance and decrease IM-induced apoptosis.3.One of the mechanisms for AF1q to function in CML is related to CD44 modulation,therefore targeting AF1q or CD44 may lead to a better therapeutic strategy for this disease.Section ?Characterization of hsa_circ₀004277 as a new biomarker for acute myeloid leukemia via circular RNA profile and bioinformatics analysisBackground:Acute myeloid leukemia?AML?represents a group of myeloid malignancies characterized by acquired genetic abnormalities in hematopoietic progenitors.Advances in molecular genetics promote the recognition of AML prognostic biomarkers,among which cytogenetic results and molecular abnormalities are considered as the most important factors.Nearly half of AML patients are found without cytogenetic abnormality,and constitutes the main part of intermediate-risk AML category.However,these cytogenetically normal AML?CN-AML?patients can be divided into different subgroups based on molecular markers such as FLT3-ITD and NPM1 mutation.Patients with FLT3-ITD mutation are in the poor-risk group,whereas patients with NPM1 mutation in the absence of FLT3-ITD or isolated>ial]elic CEBPA mutation are in the better-risk group.Currently,extensive research studies have been carried out to identify more prognostic biomarkers,focusing on various kinds of molecular signals:DNA mutations,aberrant expressions of mnicroRNA?miRNA?,and long noncoding RNA?IncRNA?.Circular RNAs?circRNAs?represent a widespread class of non-coding RNAs,which drew little attention in the past.They have a closed continuous loop configuration,with a resistance to exonucleases that makes them more stable compared with their linear counterparts.The expression of circRNA was found to be issue/developmental-stage-specific and also shown to act as miRNA sponges that regulate gene expression.Furthermore,limited evidence suggested that circRNAs are associated with cancer and may play a significant part in its pathogenesis and diagnosis.However,no data was reported regarding the roles of circRNAs in the development of AML or its response to treatment.Objective:The aim of this study is to demonstrate the expression profile of circRNAs in AML and present an AML-specific circRNA feature,especially the circRNAs related to AML risk-status.Furthermore,we intend to identify one certain circRNA from the candidates,followed by its characterization and functionally evaluation,thus offering a potential diagnostic marker and treatment target in AML.Materials and methods:1.Sample collection:Bone marrow samples from 113 CML patients and 12 control donors were obtained,from which mononuclear cells were separated.Ten samples were used for the circRNA microarray,and 115 samples were used for further investigation.2.Microarray assay:Total RNA of ten samples was isolated and treated with RNase R to remove linear RNAs,then transcribed into fluorescent complementary RNA,followed by hybridization.After incubation,the hybridized arrays were scanned and then further analyzed.3.Real-Time RT-PCR validation:Total RNA of 115 samples was reversely transcribed into cDNA.RT-PCR was conducted and a Ct method was used to analyze the gene expression level.GAPDH was used as an internal control.4.MicroRNA prediction and functional analysis:The circRNA-miRNA interaction was predicted with Arraystar's home-made miRNA target prediction software based on TargetScan and miRanda.Cytoscape was applied to build a circRNA-miRNA-mRNA/gene interaction network.The predicted gene functions in the networks were annotated using GO and KEGG pathway analysis.Results:1.Profile of circRNA expression in AML patients:In the microarray,4573 out of 13,617 circRNAs were beyond detection and selected for further analysis.Among those 4573 circRNAs,464 distinct circRNAs were found differentially expressed,in which 147 circRNAs were upregulated and 317 circRNAs were downregulated more than two-fold in CN-AML patients compared to healthy controls.Furthermore,12 dysregulated circRNAs were 10-fold more prevalent than the average of healthy controls.2.Identification of circRNA signature between better-risk and poor-risk AML patients:2.1 After further analysis between better-risk and poor-risk AML subgroups,two upregulated circRNAs?hsa_circ₀035381,hsa_circ₀049657?and three downregulated circRNAs?hsa_circ₀001187,hsa_circ₀008078,hsa_circ₀001947?were found related to AML risk-status within the whole profile.2.2 One of the well-known regulatory functions of circRNA is to serve as miRNA sponges and competitively suppress its activity.For this purpose,the five highest-ranking candidate miRNAs and their target genes for each circRNA were identified.GO and KEGG pathway analysis revealed that the target genes related to this circRNA signature participated in various biological processes,such as developmental process,regulation of cellular process and cell junction.Some vital pathways could also be influenced by the altered circRNAs,including MAPK signaling pathway,PI3K-Akt pathway,and HIF-1 signaling pathway.3.Identification of novel hsa_circ₀004277 in AML:3.1 A much lower expression level of hsa_circ₀004277 was found in newly diagnosed AML patients without prior treatment?ND group?compared to the healthy controls?ctrl group?.Meanwhile,patients achieved complete remission?CR group?after treatment restored the expression of hsa_circ₀004277,showing no difference compared to the ctrl group.However,in the relapsed-refractory patients?RE group?after CR stage,downregulated hsa_circ₀004277 expression was observed again,3.2 The receiver-operating characteristic?ROC?curve analysis showed a promising diagnostic value of hsa_circ₀004277,with an area under the ROC curve value at 0.957.3.3 WDR37,the linear isoform of hsa_circ₀004277,was found to have a lower expression in the ND group,and higher expressions in the control and CR groups similar to the result of hsa_circ₀004277.Furthermore,a positive correlation between hsa_circ₀004277 and WDR37 was observed in all the AML patients at different stages.4.Effects of chemotherapy on hsa_circ₀004277 expression in AML Patients:In matched-pair bone marrow samples acquired from AML patients at both ND and CR stage,a significant increase of hsa_circ₀004277 expression was observed in the CR stage compared with the ND stage,indicating chemotherapy could significantly restore the expression of hsa_circ₀004277.5.MicroRNA prediction and downstream Bioinformatics Analysis for hsa_circ₀004277:CircRNA-miRNA-mRNA/gene network showed that hsa-miR-13 8₅p and hsa-miR-30c-1-3p exhibited the most complicated interactions,followed by hsa-miR-892b,hsa-miR-571,and hsa-miR-328-3p.Among hundreds of mRNAs/genes identified from sequence-pairing with those five miRNAs,six of them were targeted by more than one miRNA:SH3GL2,PPARGC1A,PIP4K2C,SH2B3,ZNF275,and ATP1B4.Conclusion:1.A unique circRNA profile was provided in AML patients based on which a substantial circRNA signature was demonstrated to indicate possible involvement in AML risk-status.2.One abundant circRNA,hsa_circ₀004277,was characterized and functionally evaluated,thus offering a potential diagnostic marker and treatment target in AML.