| Background and objective:Acute lymphoblastic leukemia(ALL)is a kind of hematological malignancies in which blast cell proliferate abnormally in bone marrow and peripheral blood.Based on multiagent chemotherapy regimens,risk-stratified antileukemic therapy and good nursing,the 5-year survival rates of children with ALL have improved more than 90%.Although the overall survival rate is extremely high in recent years,more than 20% of children with ALL who achieved complete remission eventually relapsed.However,the genetic and molecular biological abnormalities involved in development of ALL were considered as the most important factor affecting long-term remission and recurrence.However,it is of great clinical significance to further explore and study the pathogenesis of childhood ALL from molecular field to improve the long-term survival rate and reduce the recurrence.MicroRNA(miRNA)is not only widely involved in the physiological and pathological activities,but also closely related to tumorigenesis and progression.At present,some studies have shown that miRNAs can also be used as early diagnostic indicators,prognostic evaluation,drug resistance and toxicity prediction.It has been confirmed that miRNA is involved in the gene expression of ALL as proto-oncogenes or tumor suppressor gene.In addition,miRNAs can also regulate the differentiation,proliferation and secretion of effector factor of immune cells in tumor microenvironment,inhibit the function of immune cells and lead to tumor immune escape to promote the tumorigensis and development of ALL.Therefore, miRNA may become an effective indicator for the diagnosis,treatment and prognosis of childhood ALL in the future.Immunosuppression and immune escape are important mechanisms of tumor progression and recurrence.Immunosuppressive regulatory T cell(Treg)is a kind of T lymphocyte subset and plays an important role in the tumor microenvironment.Treg suppresses anti-tumor immune response through multiple immune functional pathways in the tumorigenesis and progression of tumor to lead to tumor escaping from immune surveillance.The abnormal number and function of Treg participate in the occurrence and development of multiple solid tumors and hematological malignancies,and is correlated with the prognosis of tumors,which has important clinical value for the evaluation of condition and prognosis of patients.It has been found that Treg aggregates to the tumor microenvironment and peripheral sites of solid tumors through a variety of ways.However,the mechanism of Treg proliferation and recruitment in hematological malignancies remains unclear.In this study,we hypothesized that Treg plays an important role in immunosuppression and immune escape in childhood ALL,which is similar to solid tumors.The differential expression and regulation of Treg may affect the diagnosis and treatment of ALL.Previous studies have reported that abnormal expression of miRNA plays an important role in Treg induction and function.Thus,we speculated that the abnormal expression of miRNA in children with B-ALL can promote the differentiation and proliferation of Treg,leading to the increase of percentage of Treg in peripheral blood.In order to prove this hypothesis,we first need to identify the differential expression miRNA involved in regulating Treg in childhood ALL.In this study,we compared the expression of miRNA in peripheral blood mononuclear cells(PBMC)between children with B-cell acute lymphoblastic leukemia(B-ALL)and healthy children byRNA sequencing(RNA-Seq).We found that there were significant differences in gene expression between children with B-ALL and control group in proliferation,survival and immune activation regulation.Then we verified the expression of differentially expressed miRNAs by expanding the clinical sample size,and analyzed the correlation between the expression level and the percentage of Treg in peripheral blood.Our results showed that the expression level of miR-539-5p was correlated with the percentage of Treg in peripheral blood of patients.The gene enrichment analysis showed that miR-539-5p had significant differences in regulating abnormal cell proliferation and differentiation.Secondly,in order to investigate the effect of miR-539-5p regulation on Treg activation,and diagnosis and treatment of childhood ALL,we studied the correlation between the expression level of miR-539-5p and the clinical features and response to chemotherapy in children with B-ALL,and screened the differential gene bone morphogenetic protein 2(BMP2)and transforming growth factor-β(TGF-β)/Smad signal transduction pathway byRNA-Seq.Finally,the relationship between miR-539-5p and BMP2 was studied in vitro to further clarify the role of miR-539-5p and its target gene BMP2 in abnormal cell proliferation,survival and Treg activation in B-ALL.Part Ⅰ Screening of differently expressed miRNA in childhood B-cell acute lymphoblastic leukemiaObjective:The aim of this study was to compare the expression of miRNA in PBMC between children with B-ALL and healthy children byRNA sequencing(RNA-Seq),and screen differentially expressed miRNAs that may be involved in Treg,and verify the expression of differentially expressed miRNAs by expanding the clinical sample size.Methods:According toRNA-Seq requirement,each group should contain at least three samples.In this study,5 newly diagnosed children with B-ALL and healthy children were enrolled at West China Second University Hospital from September to October2019.Fresh peripheral blood was collected with vacutainer tubes containing K2 EDTA.PBMC were separated by Ficoll Hypaque density gradient centrifugation.After the totalRNA was extracted,the integrity and concentration of the samples were detected by Agilent 2100 Bioanalyzer.Library was constructed using Truseg smallRNA sample prep kit(Illumina).RNA-Seq was used to screen differentially expressed miRNAs in PBMC between children with B-ALL and healthy children.According to Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of genes and genes(KEGG)signal pathway enrichment analysis,we predicted target genes and its potential signaling pathway of differentially expressed miRNA.By expanding the clinical sample size,the expression of differentially expressed miRNAs was determined by quantitative PCR in 20 patients with B-ALL and 15 healthy children,and compared with results ofRNA-Seq to verify the expression of the differential miRNA in clinical samples.Patients were divided into low-expression group and high-expression group based on the median miRNA expression.Then,the percentage of Treg between the two groups was compared in order to find differentially expressed miRNA involved in the regulation of Treg.Results:1.A total of 1666 miRNAs were obtained fromRNA-Seq data.In the present study,107 differentially expressed miRNAs were significantly up-regulated and 132 were significantly down-regulated. 2.GO and KEGG analysis showed that differential genes were closely related to cell proliferation,survival and immune system activation,especially lymphocyte differentiation and signal transduction.The target genes enrichment of differentially expressed miRNA was mainly related to cell proliferation,survival and apoptosis.The enrichment of transforming growth factor-β(TGF-β)signaling pathway was relatively high,which was closely related to cell proliferation,differentiation and immune activation.3.Among the significantly expressed miRNAs,miRNAs with the largest fold-change were selected for further investigation.Mi R-548 ba,miR-1266-5p,miR-466,miR-551 a and miR-4484 were the most significantly up-regulated,and miR-539-5p,miR-1-3p,miR-133a-3p,miR-6718-5p,miR-11401 and miR-1250-5p were the most significantly down-regulated.The results of PCR were consistent with theRNA-Seq.4.The expression level of miR-539-5p negatively correlated with the percentage of Treg in peripheral blood of patients with B-ALL.5.The target genes of miR-539-5p were mainly enriched in the regulation of cell proliferation and differentiation,BMP2 was the most differentially expressed mRNA among the target genes of miR-539-5p.Conclusion:RNA-Seq data showed that miR-539-5p was a differentially expressed miRNA in children with B-ALL.The expression level of miR-539-5p was negatively correlated with the percentage of Treg in peripheral blood of patients with B-ALL.Part Ⅱ Correlation between miR-539-5p and clinical features of childhood acute B-lymphoblastic leukemiaObjective:On the basis of previous study,the aim of this study was to investigate the correlation between the expression level of miR-539-5p and the clinical features and response to chemotherapy of children with B-ALL.Methods:A case-control study(1:1 ratio)was conducted from September 2019 to February 2020.A total of 43 children with B-ALL who were diagnosed in the Department of pediatric hematology and oncology of our hospital were included in this study.Thirty-nine healthy children who underwent physical examination in the outpatient department of our hospital were also enrolled as the normal control group during the same period.In this study,the diagnosis of B-ALL was based on Chinese Children Cancer Group(CCCG)acute lymphoblastic leukemia(ALL)2015 protocol(CCCG-ALL-2015).The clinical characteristics of patients with B-ALL included age,gender,FAB morphology,flow cytometry immunotyping,white blood cell count,hemoglobin,platelet count,percentage of Treg,percentage of bone marrow blast cell,fusion gene,cytokines,and risk stratification.Minimal residual disease(MRD)levels on day 19 and 46 were used to evaluate the response to chemotherapy.The percentage of Treg in children with B-ALL was compared with healthy controls.Patients were divided into low-expression group and high-expression group based on the median miR-539-5p expression.The clinical characteristics and response to chemotherapy were compared between miR-539-5p low-expression group and high-expression group in patients with B-ALL.Results:1.The percentage of Treg in B-ALL group was significantly higher than that in normal control group.The percentage of Treg in patients with MRD <1% was significantly higher than that in patients with MRD ≥1% on day 19. 2.The percentage of Treg in peripheral blood of patients with miR-539-5p low-expression group was significantly higher than that in those in high-expression group.However,there were no significant differences in age,gender,FAB morphology,flow cytometry immunotyping,white blood cell count,hemoglobin,platelet count,percentage of bone marrow blast cell,fusion gene,risk stratification and cytokine(TGF-β1,IL-10 and IL-35)between low-expression group and high-expression group.3.No significant difference was found in MRD on day 19 and 46 between miR-539-5p low-expression group and high-expression group.However,two patients with low expression of miR-539-5p had positive MRD on day 19,and their MRD on day 46 were still positive.Conclusion:The percentage of Treg in patients with B-ALL was significantly higher than that of healthy children and associated with early response to therapy.Patients with low expression of miR-539-5p were likely to have a poor response to chemotherapy.Part Ⅲ Mechanism of miR-539-5p/BMP2 regulating Treg proliferation in NALM-6 cellsObjective:Our previous study found that miR-539-5p was a differentially expressed miRNA,and its expression level was associated with the percentage of Treg and response to chemotherapy in children with B-ALL.The aim of this study was to explore the role of miR-539-5p and its target gene BMP2 in regulating of Treg in NALM-6 cells in vitro.Based on the enrichment of miR-539-5p target gene BMP2 and pathway in children with B-ALL,the purpose of this study was to determine the role of miR-539-5p participating in TGF-β signaling pathway to induce Treg.We studied the mechanism of miR-539-5p regulating target gene BMP2 in the induction of Treg proliferation with differential gene BMP2 as the research target.Methods:The expression of miR-539-5p in K562,Molt4,BALL-1 and NALM-6 were detected by PCR.According to the results ofRNA-Seq,the differentially expressed genes in TGF-β signal were preliminarily screened.The level of protein and mRNA expression of key differentially expressed genes in peripheral blood of children with B-ALL were screened and verified by ELISA and PCR.The co-expression of CD19 and BMP2 in PBMC smear of patients with B-ALL was detected by immunofluorescence staining.The expression of BMP2 in K562,Molt4,BALL-1and NALM-6 were detected by PCR.The binding targets of miR-539-5p and mRNA were predicted by bioinformatics analysis and verified by the dual-luciferase reporter assay system.The expression levels of related proteins expression were detected by Western blot.We investigated the effects of miR-539-5p and BMP2 expression on the proliferation and apoptosis of NALM-6 cells.CD4~+CD25~-T cells from PBMC of healthy people were isolated and cultured by immunomagnetic beads.The co-culture system of NALM-6 cell supernatant and CD4~+CD25~-T cells was established.The percentage of Foxp3+Treg was detected by flow cytometry.The proliferation activity of CD4~+CD25~-T cells was analyzed by CFSE.The expression of TGF-β1,IL-10 and IL-35 was detected by PCR.The effect of NALM-6 cell culture supernatant combined with TGF-β1 was studied on Treg proliferation.Results:1.The expression level of miR-539-5p in different leukemia cell lines were detected by PCR.The results showed that the expression level of miR-539-5p was lower in NALM-6 cell than those in other cells.Thus,NALM-6 cell was selected for functional analysis of miR-539-5p.2.The expression level of BMP2 mRNA and protein in peripheral blood of children with B-ALL were significantly higher than those in normal controls.The expression level of BMP2 mRNA was negatively correlated with the expression of miR-539-5p.3.The co-expression of CD19+ and BMP2+ in PBMC of children with B-ALL indicated that the expression and secretion of BMP2 on CD19+ B lymphocytes in peripheral of patients were increased.4.The expression level of BMP2 in different leukemia cell lines were detected by PCR.The results showed that the expression level of BMP2 was significantly higher in NALM-6 cell than those in other cells.5.The results of dual-luciferase reporter assay system confirmed targeted relationship between miR-539-5p and BMP2.6.Overexpression of miR-539-5p could significantly inhibit the proliferation and growth of NALM-6 cell,promote the expression of Bax,and inhibit the expression of Bcl-2 and PCAN to promote the apoptosis of NALM-6 cell.7.Overexpression of miR-539-5p could reduce the expression level of BMP2 mRNA,inhibit the proliferation and promote the apoptosis of NALM-6 cell.8.BMP2 siRNA could inhibit the proliferation and promote the apoptosis of NALM-6 cell.9.BMP2 expressed and secreted by NALM-6 cell could not directly induce the differentiation of CD4~+CD25~-T cells to Foxp3+Treg.However,it enhanced the ability of TGF-β1 to induce Foxp3+Treg,and promoted the expression of TGF-β1,IL-10 and IL-35.10.BMP2 expressed and secreted by NALM-6 cell could promote the phosphorylation level of Smad1,Smad3,ERK1/2,JNK and P38 induced by TGF-β1.Conclusion:BMP2 secreted by NALM-6 cells could promote the differentiation of CD4~+CD25~-T cells to Fopx3+Treg by TGF-β1.Overexpression of miR-539-5p inhibited the differentiation of CD4~+CD25~-T cells to Fopx3+Treg by downregulating the expression and secretion of BMP2 in NALM-6 cells. |
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