Aberrant Expression And Mechanisms Of CD82 In Childhood Acute Leukemia

Part ⅠAberrant expression of CD133 and CD82 and their clinical significances in pediatric ALLBackground:Acute lymphoblastic leukemia(ALL)is the most common childhood malignancy characterized by uncontrolled clonal proliferation of lymphoid blasts with reduced capacity to differentiate into mature cells.The cure rates have increased to more than 80%in some study groups.However,about 15-20%patients eventually relapsed that it became the main obstacle to further improved treatment.Accumulated evidences have demonstrated that leukemia stem cells(LSCs),a small population of leukocytes,were responsible for relapse of ALL.Self-renewal and apoptosis are important biological properties of LSCs and can maintain the survival of leukemia cells.Moreover,most LSCs are in the GO cell cycle and are insensitive to traditional chemotherapy drugs,resulting in refractory recurrence of the disease.Therefore,the study of LSCs surface-specific molecular markers,targeted removal of LSCs,is an important direction for the current treatment of leukemia.LSCs have been shown to express some LSCs markers,such as CD90,CD96,CD117,CD44,CD123,CD133 et al.CD133,is a five transmembrane protein,which is originally identified as one marker of hematopoietic stem cells.Recent studies have implicated that CD133 has been shown to be more specific marker of hematopoietic stem cells than CD34.The expression of the CD133 antigen in acute leukemia was correlated with a more immature phenotype of the blast population and a bad prognosis.The expression of CD133 is associated with chemo-resistance.Therefore,it is important to evaluate the expression level of CD133 in clinical diagnosis and treatment.However,the reports about expression of CD133 in ALL were conflicted,especially in pediatric ALL.Some reports found high level of CD133 expression on particular cases,while others demonstrated either low level or none at all.The CD82 gene,a member of the tetraspanin superfamily(TM4SF)named KAI1.In the context of cancer,CD82 was associated with integrins on the surfaces of various tumor cells,and its expression was linked to metastasis suppression.However,in view of the specificity of tumor types,the expression of CD82 in malignant hematological diseases is very different from that in solid tumors.Studies have shown that CD82 is abnormally expressed in various types of leukemia and participates in the regulation of proliferation and adhesion of leukemia cells.Therefore,studying the expression of CD82 plays an important role in diagnosis and treatment.However,the current expression of CD82 in childhood ALL has not been reported.It has been suggested that CD82 was abundantly expressed on primitive and haemopoietic progenitor cells.It was demonstrated that CD82 regulates adhesion and survival of leukemia stem cells.However,little is known regarding the expression and roles of CD82 in BM of pediatric ALL patients and the relationship between CD82 expression and its clinical characteristics.Studies have confirmed a correlation between CD133 and CD82 expression in laryngeal squamous cell carcinoma and bladder urothelial cancer,but have not been reported in hematological diseases.This study was designed to detect the expression levels of CD133 and CD82 in childhood ALL bone marrow samples and to analyze whether there is a correlation between them.Objective:The aim of study is to explore the expression of CD133 and CD82 in pediatric ALL patients,to analyze the correlation of CD 133 and CD82 with clinical features and to explore the significance of CD82 and CD133in the treatment of ALL in children.Materials and methods:1.Sample collection and BMMCs isolation:Bone marrow samples from 37 newly diagnosed ALL children,22 children with ALL complete remission and 15 normal controls were isolated to obtain bone marrow mononuclear cells(BMMCs).2.Detection the mRNA expression of CD133 and CD82:Real-Time RT-PCR was performed to detect the expression level of CD133 and CD82 mRNA in ALL-ND,ALL-CR and controls.To explore the correlation between CD133 and CD82 by statistical methods.3.Detection of expression level of CD133 and CD82 on cell surface:The expression level of CD133 and CD82 on the surface of bone marrow mononuclear cells were detected by flow cytometry,and the relationship between them and the expression level of m RNA was analyzed.4.Detection of CD82 expression in serum:Elisa method was used to detect CD82 expression in serum of child ALL patient and controls.Results:1.Aberrant mRNA expression of CD133 and CD82 in pediatric patients with ALL.CD82 mRNA expression level was observably elevated in pediatric ALL-ND patients compared with controls.However,no significant change was found between ND and CR patients.In B-ALL,CD82 mRNA expression in ND was significantly increased compared with CR and controls.In T-ALL,CD82 expression in ND was markedly higher than that in controls.No significant difference of CD82 expression was found between CR and controls in T-ALL.In B-ALL,CD133 mRNA expression level was marginally statistically higher in pediatric ALL-ND patients compared with controls and CR patients but in T-ALL the diffe In B-ALL patients,a positive correlation was observed between CD 133 mRNA expression and CD82 mRNA expression in BM(r= 0.3174,p=0.0316)(Fig.2),but in T-ALL the correlation was not significant.2.Flow cytology detection of BMMCs showed that the proportion of CD34+,CD34+CD133+ and CD34+CD82+ in BMMCs of ND-ALL was significantly higher than that of the controls.3.The expression of CD82 in bone marrow serum detected by Elisa showed that the expression level of CD82 in the ND-ALLwas significantly higher than that of the controls,and the difference between ND-ALL and ALL-CR was not significant.4.The proportion of CD34+CD82+ cells was positively correlated with CD82m RNA,while the proportion of CD34+CD133+ cells was positively correlated with CD133 mRNA expression.5.CD133 mRNA expression in ALL patients with hyperdiploid karyotype was significantly increased compared with normal karyotype.The expression of CD133 and CD82 had no relationship with other clinical factors such as sex,age,WBC,and the infusion genes,the risk stage in ALL patients.Conclusion:1.Aberrant expression of CD133 and CD82 in childhood ND-ALL was found,and the expression level of CD133 and CD82 in child with B-ALL were deceased after complete remission,suggesting that CD133 and CD82 play a role in the development of ALL in children.2.There was a significant positive correlation between CD133 and CD82 expression levels in children with ALL,suggesting that CD133 and CD82 may synergistically affect the development of ALL in children.3.To further explore the role of CD82 and CD133 in childhood ALL may open a new way for the treatment of ALL in children.Part ⅡAberrant expression and mechanism of CD82 in childrenwith acute myeloid leukemiaBackground:Acute myeloid leukemia(AML),originated from hematopoietic stem/progenitor cells,is characterized by increased immature myeloid blasts within the bone marrow that account for approximately 20%acute leukemia in children and adolescents.With an increasing number of chemotherapy drugs being made available,AML still remains a highly fatal disease due to its significant relapse rate after standard treatment.The high relapse rate in AML is thought to be due to the lack of eradication of the leukemia stem cells(LSCs).LSCs has a strong survival advantage of self-renew and proliferate indefinitely.The interaction between LSCs and the bone marrow microenvironment allows it to escape the killing of chemotherapeutic drugs.Therefore,LSCs and bone marrow microenvironment have been studied as potential therapeutic targets,and of particular interest to researchers are the signaling pathways that control the development and survival of LSCs.CD82/KAl1,initially identified as a metastasis suppressor of some solid malignant tumors.Recently,CD82 upregulation has been identified in chemotherapy-resistant CD34+/CD38-AML cells(12),which are often responsible for disease relapse.Previous study found that CD82 regulated the hematopoietic stem and progenitor cell(HSPC)bone marrow maintenance,homing and engraftment(13).The above evidences conformed that CD82 is important to maintain the character of LSCs as a biomarker for LSCs in AML.CD82 is both expressed as a stem cell marker in LSCs expression,and as an adhesion molecule mediates the interrelationship of LSCs in the bone marrow microenvironment,plays an important role in the occurrence and development of AML.So the study of the mechanism of CD82 in AML is of great significance.However,current reports on the expression level of CD82 in children with AML are extremely rare,and the pathway and mechanism of CD82’s role in AML remain inconclusive.The Wnt signaling pathway plays an important role in the regeneration of human hematopoietic stem cells and is of great significance for maintaining normal hematopoiesis.As the core protein of Wnt signaling pathway,β-catenin can bind to the TCF/LEF transcription factor in the nucleus after cytoplasmic aggregation to form a complex to regulate the expression of Wnt-related genes,thereby participating in the regulation of cell proliferation and promoting transcription.Wnt/β-catenin is involved in the self-renewal of LSCs as an important signaling pathway,regulating the drug resistance and recurrence of leukemia.In solid tumors,CD82 regulates the survival and metastasis of cancer cells through Wnt/β-catenin signaling pathway,but the association between CD82 and Wnt/β-catenin in leukemia is rare.To study the interaction and the mechanism between CD82 and Wnt/β-catenin regulating of AML cells is of great significance for the treatment of AML.Objective:The aim of this part was to explore the expression of CD82 in AML children patients and identify the effect of CD82 overexpression on cell proliferation,cell cycle arrest and drug resistance in pediatric AML patients.Further more,we also intend to clarify the potential mechanism of CD82 in AML,thus providing the basis for a new molecular targeted therapy.Material and methods:1.Sample collection and cell isolation:bone marrow sample from 31 untreated pediatric patients with de novo AML and 25 hematologically normal age-matched BM samples were obtained,from which mononuclear cells were separated.2.Detection of CD82,β-catenin and molecules downstream of Wnt/β-catenin Real-Time RT-PCR was performed to detect the expression level of CD82,β-catenin and molecules downstream of Wnt/β-catenin from AML patients and controls.3.Thansfection of AML cell line:The AML cell line THP1 was transfected with lentivirus and the CD82 up-regulated cell lines(CD820E),the CD82 down-regulated group(CD82DR)and the stably transfected cell lines were obtained for at least two weeks puromycin screening.Real-Time RT-PCR and Western blot were used to verify the up or down-regulation efficiency of CD82.4.Cell proliferation,apoptosis,cell cycle arrest and viability assays:Transfected cells were seeded into culture plates and CCK8 assay was to examine the proliferation;the cell cycle was detected by flow cytometry.The cells were incubated with doxorubicin for 48 hours.The drug sensitivity was detected by CCK8 method and the apoptosis rate was detected by flow cytometry.5.To detect the expression of downstream molecules of Wnt/β-catenin signaling pathway after up-regulating and down-regulating CD82:western blot and immunofluorescence were used to detect the expression of β-catenin total protein,plasma protein and nuclear protein,and detect the expression level of other downstream molecules including c-myc,cyclinDl,survivn and other molecules.Results:1.Expression of CD82 in children with AML:Compared with healthy controls,the expression of CD82 mRNA in bone marrow mononuclear cells of children with AML is significantly increased,suggesting that CD82 may play a role in the development of AML.2.Correlation between CD82 expression level and clinical features of AML patients:The results showed that CD82 mRNA expression levels were significantly increased in M2 patients and patients with AML/ETO mutations.CD82 expression levels were not significantly associated with other clinical features including age,gender,and white blood cell count.3.There is a correlation between CD82 expression level and downstream molecules of Wnt/β-catenin:CD82 mRNA expression level is significantly positively correlated with β-catenin,c-myc and cyclin D1 in AML patients;AML cell line up-regulated in CD82,β The expressions of-catenin,c-myc,cyclin D1 and survivn were up-regulated.The expression of β-catenin,c-myc,cyclin D1 and survivn were down-regulated in AML cell line down-regulated by CD82.These results suggest that CD82 may regulate β-catenin and its downstream molecules in AML.4.Effect of CD82 expression on cell proliferation,apoptosis,cycle and drug sensitivity:After up-regulating CD82 expression in AML cell line THP1,CCK8 results showed that the cell proliferation rate increased and the sensitivity to doxorubicin-induced drugs decreased.Flow cytometry results showed that the apoptotic rate decreased and the proportion of cells entering the proliferative phase increased.After down-regulating CD82 expression in THPI,CCK8 results showed that the cell proliferation rate was slowed down,and the sensitivity to doxorubicin induction was enhanced.Flow cytometry showed an increase in apoptosis rate.These results indicate that CD82 can be involved in the regulation of proliferation,cycle,drug sensitivity and apoptosis of AML cells.5.CD82 participates in Wnt/β-catenin signaling pathway and enhances its activity:up-regulates the expression of CD82 in AML cell line,and finds that β-catenin is transferred into the nucleus.The nuclear accumulation of β-catenin promotes the transcription of downstream signaling molecules.Up-regulation of CD82 promotes the expression of β-catenin,c-myc,cyclinDl and survivin RNA,and promotes the expression of β-catenin and cyclinDl protein.Conclusion:1.The expression level of CD82 in AML children is higher than that in healthy controls,suggesting that CD82 plays a role in the occurrence and development of childhood AML.2.By regulating the expression level of CD82,the proliferation rate,cycle,drug sensitivity and drug-induced apoptosis rate of AML cells can be changed.It is confirmed that CD82 accelerated the proliferation of AML cells,promoted AML cells get into proliferation cell cycle,reduced the drug sensitivity and protected cells from apoptosis.3.One of the specific mechanisms of CD82 in AML is mediated by the regulation of Wnt/β-catenin signaling pathway,which provides a new way of thinking for the treatment of AML.Part ⅢStudy on the correlation between single nucleotide polymorphisms of inflammation-related genes and ALL in childrenBackground:Acute lymphoblastic leukemia is the most common malignancy in children.It was commonly seen in children aged 2-5 years.It was suggested that ALL were believed to be caused by the combination of genetic factors and environmental factors.Genomewide association studies have shown that single nucleotide polymorphism(SNP)is an important risk factor for the development of ALL in children.Clinical and experimental data demonstrate the role of chronic inflammation in the etiology of several types of cancer.More than 15%of human cancers are caused by infection,such as helicobacter pylori or hepatitis virus,which directly promotes canceration by inducing chronic inflammation.Many SNPS of cancer cytokines affect patients’ susceptibility to ALL.Therefore,to explore the correlation between the inflammatory cytokine SNP site and ALL in children may be helpful for the early detection,diagnostic evaluation and treatment monitoring of ALL.Objective:To explore the correlation between the SNP sites of inflammation-related genes and the susceptibility to ALL in children and the treatment response,so as to provide new clues for the accurate formulation of treatment programs for ALL in children.Methods:Peripheral blood samples of 165 children with ALL and 175 healthy controls in our hospital were collected and their DNA were extracted.31 inflammation-related genes SNPs were detected by multiple PCR technology.The correlation between ALL susceptibility and treatment response with the 31 SNPs were statistically analyzed.Results:1.Correlation between inflammation-related SNPs with ALL susceptibilityThe genotype and allele frequency of 31 SNPs in the case group and the normal control group were analyzed by chi-square test and Fisher’s exact test.Preliminary screening results showed that the allele frequency distribution of rs2280714 in IRF5 and rs2297630 in sdf-1 was significantly different from the genotype frequency distribution of rs4353135 in NLRP3 and rs1946518 in IL18 in children ALL and the control group.Univariate logistic regression analysis showed that 2280714 in IRP5,after adjusting for age and gender,allele G was significantly correlated with children’s ALL susceptibility(p=0.019).In NLRP3 rs4353135,compared with GG genotype,patients with TG genotype were 2.009 times more likely to develop ALL than patients with GG genotype(p=0.020).At the SNP site of SDF-1 rs2297630,compared with the genotype GG,the probability of ALL in children with genotype AG was 1.789(p=0.034).There was no significant difference in the risk between AA genotype and GG genotype.That was to say,rs2297630 genotype AG was the susceptible genotype in childhood ALL.There was significant difference between allele A and allele G at rs2297630(OR=1.799,95%CI=1.132-2.860,p=0.013).rs1946518 in IL-18,the risk of genotype TG acquiring ALL was 2.079 times higher than that of genotype TT(p=0.012).That was to say,for rs1946518 in IL-18,genotype TG was a risk gene.Multivariate logistic regression analysis showed that the risk of ALL was 1.907 times higher for NLRP3 rs4353135 genotype TG than for genotype GG(p= 0.030).The risk of ALL was 1.789 times higher for SDF-1 rs2297630 genotype AG than for genotype GG(p=0.034).The risk of ALL was 0.680 times for IRF5 rs2280714 allele A than for allele G(p=0.023).The risk of ALL was 1.762 times higher for SDF-1 rs2297630 allele A than for allele G(p=0.0 1 7).2.The relationship between inflammation-related SNPs and clinical characteristics of ALL in children(1)Correlation between inflammation-related SNPs with the risk classification The genotype distribution of IL-1β rs16944 in standard risk,、middle risk and high risk groups was statistically different(p=0.011).Comparing with the standard risk and middle risk groups,the allele G frequency of IL-1β rs16944 in high risk group was significantly higher(p=0.046,OR=3.852,95%CI=1.023-14.501).(2)On the 8th day after prednisone induced treatment,patients were divided into prednisone sensitive group and non-sensitive group based on white blood cell count of peripheral blood.It was found that the probability of chemotherapy resistance of allele C at rs1310812 was 15.833 times higher than that of T.(3)When the allele gene at rs763361 was C,the probability of MRD15(+)was 2.067 times of T.On the 33rd day,when the rs10818488 genotype was AA+AG,the probability of positive residue was 0.556 times that of GG.That was to say,genotype AA+AG with a high probability of negative MRD and a good prognosis.Conclusion:1.The SNPs of IRF5 rs2280714,SDF-1 rs2297630,NLRP3rs4353135,IL18rs1946518 were related to the susceptibility of children to ALL.2.IL-1β rs16944 SNP was correlated with ALL risk stage in children.Rs 1310812 in PTPN22 was correlated with prednisone sensitivity.rs7633631 in CD226 and rs1081 8488 in TRAF1 were related to the MRD on day 15 and day 33.