The Role Of CircRUNX1 In The Pathogenesis Of Acute Leukemia And Its Mechanism

Objective:Acute leukemia（AL）is a group of hematopoietic malignancies with high heterogeneity.The pathogenesis of AL has not yet been fully elucidated.RUNX family transcription factor 1（RUNX1）is a key regulatory factor in hematopoiesis.It forms a complex core binding factor（CBF）with co-factor CBFβ,which is involved in the generation of hematopoietic stem cells and the differentiation of myeloid and lymphoid lines.There are many types of chromosomal translocations involved in this gene,in which t（12;21）in childhood acute lymphoblastic leukemia（ALL）and t（8;21）translocation in acute myeloid leukemia（AML）are most common.The RUNX1-RUNX1T1 translocation was considered to be the driving mutation of AML and suggested a good prognosis.The germ line or somatic mutation of RUNX1 gene also plays an important role in the occurrence and prognosis of acute leukemia（AL）.In 2020,the national comprehensive cancer network（NCCN）guidelines listed Runx1 as an indicator of poor prognosis in AML patients without good prognosis and molecular genetic abnormalities.So are there any other mechanisms of this gene involved in the pathogenesis and development of AL?Circular RNA（circRNA）,are a class of closed circular,endogenous non-coding RNAs（ncRNA）,which can perform biological functions as transcription regulators,microRNA sponges and protein translation templates.As a research hotspot of ncRNA,circRNAs have been shown to be involved in the pathogenesis,drug resistance and disease progression of tumors,and can be used as therapeutic targets.In recent years,the role of circRUNX1 in the pathogenesis of solid tumors has also been reported.In 2020,a study of colorectal cancer showed that highly expressed circRUNX1（hsa_circ₀002360）could promote the growth and metastasis of cancer cells through the miR-145-5p/IGF1 signaling axis.Subsequently,Chu et al.found that circRUNX1 was highly expressed in patients with papillary thyroid carcinoma,played a carcinogenic role by regulating miR-296-3p/DDHD2 pathway and could be used as a therapeutic target this year.So far,there is no relevant research on circRUNX1 and AL.So,is circRUNX1 also involved in the occurrence and development of AL?In this study,circRUNX1 will be taken as the research object,we will start from clinical samples to study the expression of circRUNX1 in AL patients and its correlation with disease status and investigate its effects on proliferation,cycle and apoptosis of AL cells at cell level.Bioinformatics database was used to predict the function of circRUNX1 and explore the possible mechanism of its influence on the biological function of AL cells.This study aims to supplement the pathogenesis of AL and provide new possible therapeutic targets for the disease.Methods:Part Ⅰ:The expression of circRUNX1 in AL and its correlation with disease stateBack-to-back primers were designed and reverse transcription fluorescence quantitative polymerase chain reaction（RT-qPCR）was used to quantify the expression level of circRUNX1 of the bone marrow（BM）samples at diagnosis and complete remission（CR）after chemotherapy from 70 AL patients（including 55 AML cases and 15 ALL cases）and 30 BM samples of normal controls（normal donors or iron deficiency patients）from the hematology department of the second hospital of Shanxi medical university and Shanxi Provincial people’s hospital between January 2019 and November 2020.After stratifying AML patients according to their risk,the expression level of circRUNX1 in each group was compared.In addition,the expression level of circRUNX1 in BM samples of AL patients at each period（including diagnosis,CR and relapse stage）was monitored longitudinally.The expression levels of circRUNX1 were compared between BM samples from normal controls and several cell lines of acute leukemia,and the high-expressed circRUNX1 cell line was screened out.Part Ⅱ:Effects of specific knockdown of circRUNX1 on proliferation,cycle and apoptosis of THP1 cellsThree pairs of siRNAs specifically bound to the circRUNX1 junction sequence were designed,and the expression level of circRUNX1 in high-expressed leukemia cell line THP1 was knocked down by nucleofection technology.The experiment was divided into four groups:siRNA group（si-circRUNX1 group）,negative control group（si-NC group）,Mock group（transfection reagent control group）and Blank group（normal cell control group）.The siRNA with the best knockdown effect screened by RT-qPCR was used for subsequent experiments.CCK8 assay was used to detect cell proliferation at 0h,24h,48h and 72h in si-circRUNX1 group and si-NC group,and flow cytometry was used to detect cell cycle and apoptosis of THP1 cells in si-circRUNX1 group and si-NC group,and comparison was made between groups.Part Ⅲ:Functional prediction of circRUNX1 and its mechanism of promoting the proliferation of AL cellsCircatla 2.0 database was used to search the expression level of circRUNX1 in various tissues and predicted the information of RNA binding protein（RBP）binding site,internal ribosome entry site（IRES）and open reading framework（ORF）.Circbank database and circatlas 2.0 database predicted the intersection of miRNA binding to circRUNX1,Starbase database and targetscan database predicted the downstream target genes of miRNA,and combined with the literature to screen and determine the candidate miRNA and downstream target genes.RT-qPCR was used to quantitatively compare the expression levels of candidate miRNAs in primary AML patients,high-expressed cell line THP1 and normal control groups.After selecting miR-296-3p,the mRNA and protein expression levels of miR-296-3p target gene c-Myc in THP1 cells in si-circRUNX1#1 group and si-NC group were compared.CircRUNX1 full-length psiCHECK2 plasmid vector,psiCHECK2 plasmid vectors containing wild-type（WT）and mutant-type（Mut）3’-UTR binding site of target gene c-Myc were constructed by PCR and primer annealing,respectively.They were co-transfected with miR-296-3p mimic or NC mimic into 293T cells by lipoD293 transfection reagent.The verification experiment for the binding relationship between circRUNX1 and miR-296-3p were divided into circRUNX1 full-length psiCHECK2 plasmid-miR-296-3p mimic group and circRUNX1 full-length psiCHECK2 plasmid-mimic NC group.There were 4 groups of verification experiments for the binding relationship between miR-296-3p and c-Myc,which were WT-mimic NC group,WT-miR-296-3p mimic group,MUT-miR-296-3p NC group and MUT-miR-296-3p mimic group.Dual-luciferase reporter assay was used to detect renilla luciferase activity（hRluc）and firefly luciferase activity（hLuc）with hRLuc as the reporter gene and hLuc as the internal reference gene.The ratio of the two activities between each group was compared to confirm the regulatory relationship between circRUNX1 and miR-296-3p and between miR-296-3p and target gene c-MycResults:Part Ⅰ:The expression of circRUNX1 in AL and its correlation with disease state55 AML patients were divided into two groups according to whether they were accompanied by RUNX1 gene abnormality at diagnosis.CircRUNX1 expression levels were higher in BM samples of primary AML patients and ALL patients than that in normal controls（all P<0.01）.CircRUNX1 expression was higher in newly diagnosed AML patients without RUNX1 gene abnormality than that in AML patients with RUNX1 gene abnormality（P=0.0307）.The expression level of circRUNX1 in AML without RUNX1 gene abnormality and ALL patients decreased after CR（P=0.0137 and P=0.0313）and tended to the level of normal controls（P=0.0634）,while the expression level of circRUNX1 in AML patients with RUNX1 gene abnormality at CR was not significantly different from that at diagnosis（P=0.375）.The expression level of circRUNX1 in BM samples of 1 case of relapsed ALL patient at different time points of initial diagnosis and after chemotherapy was detected.The results showed that the expression level of circRUNX1 was closely related to the disease status.In addition,the expression level of circRUNX1 in high-risk and intermediate risk AML groups was higher than that in low-risk AML group,but there was no statistical difference（P=0.0556）.Compared with the BM samples of normal controls,the myeloid leukemia cell lines THP1,Dami and lymphoblastic leukemia cell line Nalm6 with high expression of circRUNX1 were screened（all P<0.001）Part Π:Effects of specific knockdown of circRUNX1 on proliferation,cycle and apoptosis of THP1 cellsThe circRUNX1 and RUNX1 mRNA expression levels in the siRNA group,the NC group,the Mock group and the Blank group were detected 24 hours after nucleofection The results showed that the knockdown effect of circRUNX1#1 was the best among the three siRNAs.Compared with the Mock group,the expression level of circRUNX1#1 of the siRNA group was significantly down-regulated（P=0.001）and the expression level of RUNX1 mRNA was not changed（P>0.05）.Comparing the cell proliferation of si-circRUNX1#1 group,si-NC group and Blank group at different time points after knockdown,the results showed that the number of cells in si-circRUNX1#1 group was lower than that in si-NC group and Blank group at 72h and the difference was statistically significant（P<0.01 and P<0.001）.Cell cycle detection results showed that the proportion of G1 phase in si-circRUNX1#1 group increased（P<0.001）,while the proportion of S phase and G2 phase cells decreased（P<0.05;P<0.01）,that is,the proliferation ability of THP1 cells decreased after the down-regulation of circRUNX1.Annexin V and 7-AAD double staining assay were used to detect the apoptosis of si-circRUNX1#1 group and si-NC group,and the results showed that the proportion of early apoptotic cells in si-circRUNX1#1 group was only slightly higher than that in si-NC group（1.0%vs 0.8%）,indicating that circRUNX1 had only a weak effect on apoptosis（P>0.05）Part III:Functional prediction of circRUNX1 and its mechanism of promoting the proliferation of AL cellsCircatla 2.0 database showed that the expression level of circRUNXl in thymus,BM and spleen was higher than that in other tissues.A total of 32 RBPs were predicted to bind to circRUNX1,among which U2AF2 had the most binding sites,followed by IGF2BP2 and EIF4A3.In addition,four IRESs and two ORFs were predicted,suggesting that it has the potential to translate encoded proteins.Circbank database and circAtlas 2.0 database jointly predicted 20 miRNAs that bind to circRUNX1,miRanda and Targetscan websites predicted the downstream target genes of miRNA,and selected miRNAs hsa-miR-345-5p,hsa-miR-296-3p which play a role in tumor suppression in malignant tumors and its downstream target genes AKT and c-Myc based on literature reports to explore the mechanism of ceRNA.Comparing the expression levels of miR-296-3p and miR-345-5p in the BM samples of patients with de novo AML patients and normal controls and the circRUNX1 high-expressed cell line THP1,the results showed that the expression level of miR-296-3p and miR-345-5p in AML patients was lower than that of normal controls（P=0.0047;P=0.0301）.The expression level of miR-296-3p in THP1 cells was also significantly lower（P=0.0007）,while the expression level of miR-345-5p was only slightly lower than that of normal control BM samples（P=0.3734）.Therefore,miR-296-3p was selected for further researchIn this study,full-length reporter vectors of circRUNX1 and wild-type and mutant-type plasmid vectors of miR-296-3p predicted binding sites in c-Myc 3’-UTR region were successfully constructed.The circRUNX1 full-length psiCHECK2 plasmid was co-transfected into 293T cells with miR-296-3p mimic or mimic NC.HRLuc was used as the reporter gene,and HLuc was used as the internal reference gene.After 48 hours,the luciferase activities of HRLuc and HLuc in each well were detected and showed that miR-296-3p can inhibit the activity of the reporter gene hRluc by combining with circRUNX1.293T cells were co-transfected with wild-type and mutant-type plasmid vectors of miR-296-3p prediction binding sites in c-Myc 3’-UTR region and miR-296-3p mimic or mimic NC,and reporter gene detection was performed after 48 hours.Results showed that the ratio of the two luciferase activities of WT-miR-296-3p mimic group was significantly lower than that of WT-mimic NC group（P=0.024）,and there was no statistical difference between the Mut-mimic NC group and Mut-miR-296-3p mimic group（P=0.434）.This indicates that miR-296-3p has a direct target regulation relationship with c-Myc gene.Then,the mRNA and protein expression levels of c-Myc in si-circRUNX1#1 group and si-NC group were further compared,and both RT-qPCR and western blot results showed that the mRNA（P=0.0039）and protein expression levels of c-Myc in si-circRUNX1#1 group were lower than those in si-NC group（P=0.0053）Conclusion:1.The expression level of circRUNX1 in BM samples of AML and ALL patients was higher than that of normal controls and correlated with the disease state of AL patients.2.After knocking down circRUNX1,the proliferation of THP1 cells decreased,the proportion of G1 phase increased,the proportion of apoptotic cells only slightly increased indicating that circRUNX1 could promote proliferation but had only a weak effect on cell apoptosis3.The database predicted that circRUNX1 had 32 RBP binding sites,4 IRESs and 2 ORFs,suggesting its potential for gene regulation and protein coding4.The expression levels of miR-296-3p and miR-345-5p in BM samples of newly diagnosed AML patients were lower than those of normal controls.For THP1 cells,only the expression level of miR-296-3p was lower than the BM samples of normal controls5.After siRNA-targeted knockdown of circRUNX1,the mRNA and protein expression levels of c-Myc in THP1 cells were down-regulated6.CircRUNX1,miR-296-3p and c-Myc have a direct targeted regulatory relationship,that is,circRUNX1 can regulate the high expression of c-Myc through sponge miR-296-3p to promote the proliferation of AL cells.