Cd20 In Chronic Lymphocytic Leukemia Cells In The Experimental Study Of Low Expression Mechanism

Part ⅠThe prognostic value of CD20percent and fluorescence intensity inchronic lymphocytic leukemiaObjectives: Heterogeneity of CD20expression was existed in chronic lymphocyticleukemia (CLL), so we explore the prognostic significance of CD20expression inChinese patients with CLL.Methods: One hundred and seventy-two consecutive Chinese patients with CLLwere enrolled from January2003to December2011. Blood samples collected atdiagnosis were available for CD20, ZAP-70and CD38analyses.Multi-parameter flowcytometry was used to detect the expression of CD20on CD19+cells. Molecularcytogenetic aberrations were detected by florescence in situ hybridization (FISH).p53mutation and immunoglobulin variable heavy chain (IGHV) mutation status wereanalyzed by polymerase chain reaction (PCR) and direct sequencing.Results: In172CLL patients, the median expression percent of CD20was97.82%(range,0–100), and the median mean fluorescence intensity (MFI) of CD20on CLLcells was731.45(rang,0–9071.90). The percentage of CD20+cells in cases of IGHVmutated groups was higher than unmutated groups (mean,92.1%vs.80.4%,P<0.001). There were no difference in the MFI of CD20+cells in cases of eachprognostic factor group. The data of percentage of CD20using a receiver operatingcharacteristic plot (ROC) reflects separation between the two IGHV groups, with anarea under the curve (AUC) of0.661(95%CI:0.569to0.753). At the cutoff value ofpredicting IGHV mutation is60.30%of CD20percentage, the sensitivity and thespecificity were90.00%and38.46%. Patients whose percentage of CD20antigen above60.30%had a better treatment free survival (TFS)[Hazard Ratio (HR),0.452,95%CI:0.232to0.884, P=0.020]. Percentage and MFI of CD20were the variablesnot associated with TFS by multivariate Cox regression analysis (P<0.05).Conclusions: Expression levels of CD20is one of the prognostic markers of CLL,high level of CD20expression in CLL appears to be associated with a good prognosis,but not independent prognostic factor. PartⅡStudy on the relation between single nucleotide polymorphisms ofCD20and low expression of CD20in chronic lymphocytic leukemiacellsObjectives: Rituximab (RTX) is now widely used in clinical practice. Some CLLpatients with primary or secondary resistance to the drug, the mechanism was not yetclear. This research focused on the relationship between CD20coding regionmutations with CD20expression.Methods: Primers were designed for the CD20gene coding region.CD20wasamplified by PCR in92cases of newly diagnosed CLL patients and200healthydonors. Relative quantitative analysis of CD20mRNA was conducted in peripheralblood specimens of CLL patients. Proportions of CD20expression and fluorescenceintensity were detected by flow cytometry.Results: Exon-3c.246C>T (rs17155019) was present in3.26%(3/92) of newlydiagnosed CLL patients. Exon-4c.632C>T (rs2070770) was present in8.69%(8/92) of newly diagnosed CLL patients. There were not found of any abnormal in theremaining exons. The frequency of C/C genotype of rs17155019and rs2070770wassignificantly higher than the normal control population,96.74%and93%;91.31%and81.00%, respectively (P<0.01). C/C genotype of two SNP may be susceptiblegenotype of CLL. No statistically significant difference in the CD20mRNA,proportion and intensity of CD20expression was found in the different genotypes oftwo polymorphic loci (P<0.05).Conclusions: There was no correlation between low expression of the CD20andmutation of CD20gene coding region. Part ⅢStudy on the relationship between microRNA and low expression ofCD20in chronic lymphocytic leukemia cellsObjectives: microRNA has been shown to play an important role in the development,apoptosis, proliferation and differentiation of organisms. It is mainly through thecomplete or incomplete matching with3’UTR of the target gene, which leaded to thedegradation of the target gene mRNA or inhibiting its translation. The purpose of thisexperiment was to study the relationship between miRNA and CD20expression.Methods: To found potential miRNA which might regulate CD20expression, threedatabases, hsa-miR-1, hsa-miR-221, hsa-miR-499-3p, hsa-miR-576-5p, hsa-miR-632and hsa-miR-708were chosen. In vitro chemical synthesis of six miRNA mimicswere transfected to primary cells of13patients with newly diagnosed CLL bylipofectamine2000and cultured24h. Relative quantitative analysis of CD20mRNA was conducted in peripheral blood specimens of CLL patients. Proportions of CD20expression and fluorescence intensity were analysed by flow cytometry.Results: The expression levels of hsa-miR-1, hsa-miR-221, hsa-miR-499-3p,hsa-miR-576-5p, hsa-miR-632and hsa-miR-708were detected by RT-PCR,suggesting transfection efficiency was acceptable. CD20transcriptome and proteinlevels were detected in transfected with miRNA mimic and negative control (NC)group, respectively. No statistically significant difference was found between miRNAmimic groups and control group (P<0.05).Conclusions: The target of hsa-miR-1, hsa-miR-221, hsa-miR-499-3p,hsa-miR-576-5p, hsa-miR-632and hsa-miR-708does not include CD20. Lowexpression levels of CD20in CLL cells may not be regulated by microRNA. Part ⅣStudy on the relations between methylation levels of CD20promoterand low expression of CD20in chronic lymphocytic leukemia cellsObjectives: Epigenetics has a pivotal role in many pathological processes of cancer.Low expression of the CD20antigen in CLL may be associated with epigeneticregulation. This study focused on the relationship between histone acetylation,promoter methylation and expression of CD20.Methods: Primary CLL cells were treated with1mM,2mM, and4mM of VPA(Valproic acid), which was histone deacetylase inhibitors. After cultured24h and48h, relative quantitative analysis of CD20mRNA was conducted by PCR; Proportion of CD20expression and fluorescence intensity were detected by flow cytometry. ForCLL primary cells treated with5-Aza-dC (100μM) for24h,48h and96h, relativequantitative analysis of CD20mRNA was conducted by PCR; Proportion of CD20expression and fluorescence intensity were detected by flow cytometry. CD20promoter region was analysed by bioinformatics software. Methylation analysis ofCD20promoter region in CLL cells by Bisulfite genomic sequencing (BSP),including: genomic DNA with treated by bisulfite; Three pairs of primers weredesigned,6CpG in CD20promoter region were amplified by PCR.; Tapping andpurification of PCR products, TA cloning, picking10clones for sequencing.Results: CLL cells were treated with1mM VPA for24h, CD20mRNA levelsincreased significantly, but no significant increase in the level of CD20protein. CD20mRNA level can obviously increase after24h of5-Aza-dC treatment; The proteinlevel can obviously increase after48h. There were partially methylated in six CpGby BSP,28%(14/50),24%(12/50),8%(4/50),20%(10/50),6%(3/50) and16%(8/50), respectively. In one patient, the CD20expression was significantly increased by5-Aza-dC, the CD20promoter methylation ratio was11.67%(7/60). No significantcorrelation between the proportion and intensity of CD20expression and promotermethylation was detected.Conclusions: Epigenetic mechanisms may be associated with low CD20expressionin CLL; Demethylation drugs can increase the expression of CD20in CLL cells.