| Background:Leukemia is a malignant clone disease which occurs in a haematopoietic stem cell.Leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to uncontrolled proliferation,dysdifferentiation,blocked apoptosis and other mechanisms,infiltrate other non-hematopoietic tissues and organs,and inhibit normal hematopoietic function.Clinically,anemia,hemorrhage,infection and fever,liver,spleen,lymph node enlargement and bone pain can be seen to different degrees.According to the different disease types of leukemia patients,different treatment methods should be adopted.Currently,some types of leukemia can be cured.However,due to the severe condition and high mortality rate,there is no effective treatment method for acute B-lymphoblastic leukemia.Therefore,new treatment methods and new drugs still need to be developed.CircularRNA(circRNA)is a non-codingRNA molecule with a closed ring structure,which is produced by alternative splicing of pre-mRNA and does not have the 5 'and 3'ends of traditional linearRNA.It is widely expressed in human cells and has stability and conservation.With the deepening of its research,it is found that circRNA have many functions: 1.It can bind to a specific miRNA or a group of miRNAs,thus inhibiting the function of miRNAs and realizing the regulation of gene expression;2.It can regulate the linear splicing of pre-mRNA,and both circRNA and mRNA come from the same pre-mRNA alternative splicing,which means that the production of circRNA may affect the splicing process of the remaining sequences;3.circRNA can regulate the transcription and translation of genes.Some studies have found that some circRNA,which retains a part of exon sequence from linear genes,can recruitRNA polymerase II into the promoter region of linear genes by acting on U1 component in splicing complexes,thus enhancing the expression of the genes.Other studies have found that circRNA contains the start codon of the original gene in the process of splicing and cyclization,which may cause mRNA homologous to circRNA to escape transcription,thus regulating gene expression at the translation level;4.CircRNA can interact with proteins and regulate its targeting function toRNA molecules by binding withRNA-binding protein(RBP).In addition,Du et al.found that circRNA can form complexes with kinase inhibitor protein(p21)and cyclin-dependent kinase 2(CDK2),thus inhibiting the promotion of cell division by CDK2 and hindering cell cycle progression.5.circRNA has translation function.Recent in vitro studies have shown that circRNA has the potential to encode proteins.For example,experiments in Drosophila clearly show that circRNA are associated with translational polyribosomes.Since circRNA possesses such characteristics,it has attracted more and more attention and become a potential research and development direction of new drugs.Untill now,circRNA has been extensively studied in gastric cancer,lung cancer,liver cancer,thyroid cancer,pancreatic cancer,colorectal cancer,breast cancer,cervical cancer and osteosarcoma.Meanwhile,circRNA also plays an important role in hematologic diseases.It has been reported that in AML: PML can promote cell proliferation through AKT pathway;circFOXO3 can affect cell apoptosis;circNPM1 can influence differentiation and promote the occurrence of leukaemia;circKLHL is closely related to the prognosis of patients;circVIM can be seen as a tumor biomarker.In ALL: circAF4 can promote the occurrence and development of leukemia;circPAX5 can promote B cell maturation.In CLL: circRPL15 can be seen as a biomarker,but there are few studies on acute B lymphoblastic leukemia.Therefore,screening circRNA with different expression and exploring its expression mechanism are of great significance for the treatment of acute B-lymphoblastic leukemia.Objective: The circRNA expression spectrum of human normal B lymphoblastocyte line HMy2.CIR and human acute B-lymphoblastic leukemia cell line Ball-1 was established;the circRNA with different expression was screened;the potential molecular mechanism of differential expression of circRNA was analyzed.Method: 1.TotalRNA was extracted from human B lymphoblastic cell lines(HMy2.CIR)and human acute B lymphoblastic leukemia cell lines(Ball-1);after the circRNA were enriched and pretreated,the sequencing library was constructed;the library was denatured into single-stranded DNA molecules,captured on Illumina flowcell,and amplified into cluster in situ.The circRNA expression profiles in the two cell lines were obtained by high-throughput sequencing technology.2.Bioinformatics was used to analyze the expression profile of circRNA in HMy2.CIR and Ball-1 cell lines by.Taking HMy2.CIR as normal control,7 circRNA with low expression and significant difference in Ball-1 were selected for further study.3.QRT-PCR was used to detect the expression levels of the above seven circRNA in HMy2.CIR and Ball-1 cell lines and bone marrow of patients with acute B-lymphoblastic leukemia and non-acute B-lymphoblastic leukemia.Finally,circRNA(XLOC?011567)with the most significant difference was selected for the next study.4.Bioinformatics method was ultilized to predict the target gene of XLOC?011567 and analyse the possible signaling pathway involved with XLOC?011567 so as to analyse and forecast the ceRNA regulatory network of XLOC?011567.5.Plasmid was used to achive the overexpression of XLOC?011567 in Ball-1 cells.CCK8 method was applied to detect cell proliferation;Annexin V-PI double staining was ultilized to observe cell apoptosis;Western-blot was adopted to study the expression of key proteins of possible signaling pathway involved with XLOC?011567.6.In 293 T cells,luciferase reporter gene system was constructed to verify the interaction between XLOC?011567 and miR?6760?5p,and between miR?6760?5p and TP53I11.7.We investigated TP53I11 level in XLOC?011567 overexpressed and miR?6760?5p overexpressed Ball-1 cells,as well as the TP53I11 level in XLOC?011567 and miR?6760?5p co-transfected Ball-1 cells.Results:1.The circRNA expression profiles in human acute B lymphoblastic leukemia cell lines(Ball-1)and human normal B lymphoblastoid cell lines(HMy2.CIR)were obtained by sequencing.14720 kinds of circRNA were detected in HMy2.CIR,and 7516 kinds of circRNA were detected in Ball-1.Compared with HMy2.CIR,2387 kinds of circRNA were highly expressed in Ball-1(of which 1318 were not reported in the literature),and 12561 kinds of circRNA were low or not expressed in Ball-1(of which6613 were not reported in the literature).2.Compared with HMy2.CIR,circ?SPECC1,LOC105370956(XLOC?011567),circ?ARHGAP10,and circ?RIMBP2 were lower expressed in the Ball-1 cell line.3.MiRanda and PITA software jointly predicted that XLOC?011567 may combine miRNA,with a total of 50 miRNA that may bind.Mi Randa,targetscan,and PITA software predicted the 50 miRNA-acting target genes described above,taking a total of 133,118 potential target genes.4.The cell proliferation rate of Ball-1 cells with XLOC?011567 overexpression decreased while the early apoptosis rate increased.In addition,the expression of P-P38,P-ERK1/2,P-C-Raf,P-Gsk-3? protein increased;the expression of ERK1/2,P-AKT(Ser473),P-AKT(Thr308)protein expression decreased;the expression of JNK,P38,AKT,P-PTEN and P-PDK1 protein had no significant change.5.We proved that miR?6760?5p interacts with XLOC?011567 and TP53I11 by luciferase reporter system in 293 T cells.6.The expression of TP53I11 is increased in XLOC?011567 overexpressed and deceased in miR?6760?5p overexpressed Ball-1 cells,respectively.The applicationof miR?6760?5p mimics reduced TP53I11 augmentation caused by XLOC?011567overexpression.Conclusions:1.XLOC?011567 is low expressed in Ball-1 and bone marrow of patients with acute B lymphoblastic leukemia.2.Overexpression of XLOC?011567 can inhibit cell proliferation and promote early apoptosis in human acute B lymphoblastic leukemia cell line(Ball-1).3.PI3K/AKT,MAPK/ERK and MAPK/P38 pathways were altered in Ball-1 cell line compared with normal B lymphoblastoid cell line(HMy2.CIR).In HMy2.CIR,the expression of XLOC?011567 can affect the phosphorylation of AKT,thus affecting cell proliferation and apoptosis.4.XLOC?011567 can affect cell proliferation and apoptosis by the mechanism of XLOC?011567/hsa?miR?6760?5p/TP53I11. |
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