Hsa?circ?0097878 Regulates CD27 And IL4R Through MiR-6773-3p And Participates In The Pathogenesis Of Acute Lymphocytic Leukemia

Objective:Leukemia is a kind of malignant clonal disease of hematopoietic stem cells.Leukemia cells proliferate and accumulate in the bone marrow and other hematopoietic tissues.They also infiltrate other tissues,and organs,while inhibiting normal hematopoietic function.The clinical symptoms are anemia,bleeding,infection,fever,enlargement of the liver,spleen,lymph nodes and bone pain.In recent years,the incidence rate of leukemia has gradually increased,ranking first in young people with malignant diseases^[1].Acute lymphoblastic leukemia is the most common in children.Pathology showed that abnormally differentiated lymphocytes gathered in the bone marrow and inhibited the production of normal blood cells^[2].At present,based on bone marrow transplantation,traditional combined chemotherapy and other technologies,the application of drug targeted therapy,cellular immunotherapy and other technologies have significantly improved the therapeutic effect of all,but frequent recurrence and drug resistance are still difficult problems to combat^[3].Therefore,exploring the pathogenesis of acute lymphoblastic leukemia and finding molecular markers related to prognosis in order to improve the cure rate and prognosis has always been an important topic in leukemia research.Circular RNA(circ RNA)is a new type of noncoding RNA different from traditional linear RNA.Its closed circular structure can escape the degradation of RNase R and is more stable than linear RNA.circ RNAs can affect gene expression regulation as mi RNA sponge^[3],RNA binding protein^[4],transcription and translation regulator.And it plays an important role in cancer development,metastasis and treatment response^[5].Circ RNAs have species conservation in sequence,tissue specificity and timing specificity in expression mode,and high stability in structure,so they have the potential to become disease molecular markers^[6].However,there are few studies on the functional role of circ RNA in the diagnosis and treatment of acute B-lymphocyte leukemia.In the early stage,our research group used high-throughput sequencing technology to detect the circ RNA expression profiles of Ball-1 and HMy2.CIR cell lines,and screened the hsa?circ?0097878 differentially expressed in Ball-1.This study verified that through experiments there is a correlation between hsa?circ?0097878 and leukemia,further exploring that hsa?circ?0097878 plays the role of mi RNA sponge,participates in the mechanism of leukemia occurrence and development through ce RNA mechanism,and it looks for key molecular markers to provide the theoretical basis for effective diagnosis and treatment of leukemia.Methods:1.Correlation analysis between hsa?circ?0097878 and leukemia.1.1 Detection of the expression of hsa?circ?0097878 in leukemia cell lines and clinical bone marrow samples was achieved by RT q PCR.The construction cell line of hsa?circ?0097878 over-expression by cell transfection,CCK-8 and flow cytometry were used to detect the effects of hsa?circ?0097878 on the proliferation and apoptosis of Ball-1 cells.1.2 RNA Seq method was used to detect the gene expression profile of Ball-1 cell line before and after over-expression of hsa?circ?0097878.Bioinformatics software such as GO and KEGG were used to analyze the biological function and mechanism of hsa?circ?0097878.2.hsa?circ?0097878 regulates the expression of IL4R and CD27 through mir-6773-3p and participates in the pathogenesis of acute lymphocytic leukemia.2.1 Bioinformatics analysis and nucleocytoplasmic separation experiments verified the subcellular localization of hsa?circ?0097878.2.2 The mi RNA and mi RNA downstream target genes bound to hsa?circ?0097878were predicted by bioinformatics software.The hsa?circ?0097878-mi RNA-m RNA regulatory network was constructed.Using the GEO database,two data sets containing clinical sample data resources were selected for differential gene analysis,PPI network was constructed,hub gene was screened,and gene correlation analysis with ce RNA network was carried out to determine the key genes in ce RNA network.2.3 The regulatory mechanism of hsa?circ?0097878 on mi R-6773-3p,CD27 and IL4R and the function of mi R-6773-3p in Ball-1.After transfection of mi RNA mimics or inhibitors,the molecular mechanism of interaction and regulation between hsa?circ?0097878 and mi R-6773-3p,and CD27?IL4R binding sites were verified by double luciferase reporter gene,RT q PCR,ELISA and Western blot.The effects of mi R-6773-3p on proliferation and apoptosis of Ball-1 cells were detected by CCK-8 and flow cytometry.Result:1.Correlation analysis between hsa?circ?0097878 and leukemia.1.1 The expression level of hsa?circ?0097878 decreased significantly in leukemia patients and leukemia cell lines,and insignificantly expressed in Ball-1 cell lines.hsa?circ?0097878 can stably exist in cells,resist RNase R treatment,is not degraded easily,and has ring characteristics.Overexpression of hsa?circ?0097878 can promote Ball-1 cell apoptosis and inhibit cell proliferation.1.2 The RNA differential gene expression profile of Ball-1 cell line before and after over-expression of hsa?circ?0097878 was established.434 differentially expressed genes were found(P-value<0.05,|log₂FC|?1.5),of which 259 were up-regulated and 175were down regulated.In GO analysis,DEGs are mainly concentrated in the immune system,signal transduction,signal molecule interaction and other related pathways.KEGG enrichment analysis showed that hsa?circ?0097878 was involved in NF-?B signaling pathway and cytokine cytokine receptor interaction signaling pathway regulation mechanism.2.The hsa?circ?0097878 regulates the expression of IL4R and CD27 through mi R-6773-3p and participates in the pathogenesis of acute lymphocyte leukemia.2.1 Bioinformatics predicted that 5 mi RNAs are most likely to bind to hsa?circ?0097878,and 1083 overlapping target genes were predicted by Target Scan and mir RDB.After screening with DEGs obtained from RNA Seq before and after over-expression of hsa?circ?0097878 in Ball-1 cell line,15 m RNAs were obtained.The hsa?circ?0097878-mi RNAs m RNA regulatory network was constructed.2.2 The data sets GSE76349 and GSE34861 containing B-ALL clinical data resources were screened.The common differential genes in the two data sets,the differential genes of RNA sequencing expression profile before and after overexpression hsa?circ?0097878 and the predicted target m RNA were intersected with Venn diagram.Finally,the common gene IL4R was obtained.IL4R is the target gene of mi R-6773-3p.Through the functional analysis of the protein interaction network encoded by mi R-6773-3p target gene,the genes related to proliferation and apoptosis were screened,and IL4R and CD27 were determined to be the key genes in the ce RNA network.2.3 The hsa?circ?0097878 was mainly located in the cytoplasm.Bioinformatics analysis and double luciferase reporter gene verified the targeting relationship between mi R-6773-3p and hsa?circ?0097878.IL4R and CD27 were the direct targets of mi R-6773-3p.mi R-6773-3p can positively regulate the proliferation of Ball-1 cells and inhibit apoptosis.The rescue experiment confirmed that the biological function of hsa?circ?0097878 affecting the proliferation and apoptosis of Ball-1 cells was realized through the targeted regulation of mi R-6773-3p.2.4 The mi R-6773-3p negatively regulates the expression of IL4R and CD27 in leukemia cells.IL4R and CD27 were positively regulated by hsa?circ?0097878 in leukemia cells.The rescue experiment confirmed that mi R-6773-3p could reverse the expression of IL4R and CD27 by hsa?circ?0097878.The regulatory effect of hsa?circ?0097878 on IL4R and CD27 is mediated by mi R-6773-3p.Conclusion:1.The hsa?circ?0097878 is significantly underexpressed in leukemia cell lines and bone marrow samples of leukemia patients.Overexpression of hsa?circ?0097878 can promote Ball-1 cell apoptosis and inhibit cell proliferation,indicating that hsa?circ?0097878 is related to leukemia and may be an important biological regulatory target in the occurrence and development of leukemia.2.The hsa?circ?0097878 may directly bind to mi R-6773-3p in leukemia cells,regulate the expression level of downstream IL4R and CD27 through mi R-6773-3p,resulting in regulation of the biological characteristics of leukemia cell proliferation and apoptosis,so as to participate in the pathogenesis of acute lymphocyte leukemia.