Expression Of NDRG2 Gene In Chronic Lymphocytic Leukemia And Its Regulatory Mechanism

Objectives: N-Myc downstream regulated gene 2(NDRG2)is a member of NDRG family,which plays an important role in the regulation of cell proliferation,differentiation and apoptosis and is closely related to the occurrence and development of multiple tumors.The purpose of the study was to investigate the expression of NDRG2 in chronic lymphocytic leukemia(CLL)and explore its clinical significance.Methods: m RNA levels of NDRG2 were quantified using real-time quantitative reverse transcription-polymerase chain reaction(q RT-PCR)in 102 CLL patients and 40 healthy donors.The expression level of relative m RNA was analyzed by 2(-ΔCT)method.Flow cytometry(FCM)was used to detect the expression of CD38 and ZAP-70.The p53 mutations and immunoglobulin variable heavy chain(IGHV)mutation status in CLL patients were detected by polymerase chain reaction(PCR)and direct sequencing.Cytogenetic abnormalities were detected by interphase florescence in situ hybridization(FISH).Statistical analysis was performed by SPSS(version 20.0)and Graph Pad Prism(version 5.0).The difference of target gene m RNA expression between groups with different prognostic factors was described using the Mann–Whitney U test.Overall survival(OS)and time to first treatment(TTT)were estimated by the Kaplan-Meier method and results were compared using the log-rank test.P values less than 0.05 were considered significant.Results: Of the 102 patients and 40 controls,the relative expression level of NDRG2 spanned from 0 to 0.0467(median was 0.0017)and from 0.0028 to 0.0343(median was 0.0044),respectively.NDRG2 expression level was significantly lower in CLL samples(P<0.001).NDRG2 expression was significantly higher in patients with early Binet stages than advanced stages(P=0.022),CD38 negative than positive(P=0.013),no p53 mutations than p53 mutations(P=0.026)and without del(17p13)than with del(17p13)(P=0.005).The median TTT was 59 months and 21 months in the high and low group of NDRG2 expression,respectively.Significant difference was found between two groups in TTT(P=0.019).And with the median follow up time of 40 months(range: 6-125 months),the OS was 92.3% and 18.9% in the high and low group of NDRG2 expression,respectively.Significant difference was also found between two groups on OS(P=0.024).Conclusions: The low level expression of NDRG2 was observed in CLL.NDRG2 m RNA expression was correlated with many clinical features and had prognostic significance.Objectives: The aim of the current study was to investigate the relationship between the NDRG2 gene promoter methylation and its low expression in chronic lymphocytic leukemia(CLL).Methods: A total of 40 untreated CLL patients and 10 matched controls were collected.DNA samples were extracted from peripheral blood or the isolated lymphocytes.Methylation-specific polymerase chain reaction(MSP)using primers specific to sequences corresponding to either methylated or unmethylated DNA sequences was performed.Methylation results were validated by gene sequencing.Flow cytometry(FCM)was used to detect the expression of CD38 and ZAP-70.The p53 mutations and immunoglobulin variable heavy chain(IGHV)mutation status in CLL patients were detected by polymerase chain reaction(PCR)and direct sequencing.Cytogenetic abnormalities were detected by interphase florescence in situ hybridization(FISH).Statistical analysis was performed by SPSS(version 20.0).The difference of target gene m RNA expression between groups was described using the Mann–Whitney U test.P values less than 0.05 were considered significant.Results: Methylation of NDRG2 promoter was found in 4(10%)of the 40 CLL patients,but not in the healthy controls.These results were identified by directly sequenced.Those who were unmethylated in NDRG2 gene reveal a single nucleotide substitution of C→T,but the others had no change on single nucleotide sequence.NDRG2 expression was detected in 21 of 40 CLL patients,including 4 showed promoter methylation in NDRG2.The relative expression level of NDRG2 spanned from 0.0003 to 0.0166(median was 0.0010)and from 0 to 0.0352(median was 0.0019)in methylation and unmethylation of NDRG2 promoter of CLL patients,respectively.However,no statistical difference was observed in the two groups(P=0.965).Conclusions: Methylation of NDRG2 promoter occured in the CLL patients,but the methylation status of NDRG2 promoter was not dominant and there was no association between the promoter methylation of NDRG2 gene and its low expression in CLL.Objectives: Micro RNA(miRNA,miR)is mainly through the complete or incomplete matching with 3’UTR of the target gene,which leaded to the degradation of the target gene m RNA or inhibiting its translation of the target gene.It plays a role of tumor suppressor genes or oncogenes in tumorigenesis.The purpose of the study was to study the relationship between miRNA and NDRG2 expression in chronic lymphocytic leukemia(CLL).Methods: Bioinformatics miRNA databases were used to identify NDRG2-related miRNAs.The common conserved miRNAs with higher scores were selected and further validated by dual-luciferase reporter gene assay.We performed a renilla-luciferase assay with a modified expression p EZX vector containing the complete 3’ untranslated regions(UTR)region of NDRG2 cloned in the 3’UTR region of dual luciferase gene.For dual-luciferase assay the miRNAs or negative controls(NC)were transfected in the 293 T cell line together with p EZX vector using lipofectamine 2000,and luminescence was read after 24 h.Further validation of the miRNA target was performed by real-time quantitative reverse transcription-polymerase chain reaction(q RT-PCR)and Western blot(WB)in 11 primary CLL cultured cells.Annexin/PI staining flow cytometry(FCM)observed the percentage of cell apoptosis.FCM was used to detect the expression of CD38 and ZAP-70.The p53 mutations and immunoglobulin variable heavy chain(IGHV) mutation status in CLL patients were detected by PCR and direct sequencing.Cytogenetic abnormalities were detected by interphase florescence in situ hybridization(FISH).Statistical analysis was performed by SPSS(version 20.0)and Graph Pad Prism(version 5.0).The difference of miRNA and NDRG2 expression as well as apoptosis rate between groups was described using the Paired-Samples T Test.P values less than 0.05 were considered significant.Results: The bioinformatics analysis identified six micro RNAs as possible regulators of NDRG2(hsa-miR-29 a,hsa-miR-29 c,hsa-miR-28-5p and hsa-miR-650).The dual-luciferase assay confirmed that the mimics of hsa-miR-28-5p and hsa-miR-650 suppressed the activity of NDRG2.The analysis of these miRNAs showed that hsa-miR-28-5p and hsa-miR-650 were significantly downregulated after transfection with those miRNA inhibitors(P=0.009 and P=0.019,respectively)compared with NC.And NDRG2 level deteceted by q RT-PCR and WB was significantly higher in group of transfection with hsa-miR-28-5p inhibitor and hsa-miR-650 inhibitor compared with NC.Transfection of hsa-miR-28-5p inhibitor and hsa-miR-650 inhibitor into CLL cells from patients without p53 deletion led to significant increases in apoptosis compared to NC.However,no increased apoptosis rates of the CLL cells were found in p53 deletion patients after transfection with the above miRNAs inhibitor.Conclusions: The low expression of NDRG2 was likely to be correlated to the regulation of hsa-miR-28-5p and has-miR-650 in patients with CLL.Moreover,inhibition of hsa-miR-28-5p and hsa-miR-650 may contribute to CLL cells apoptosis by increasing NDRG2 activity in patients without p53 deletion.