| It is well known that acute leukemia (AL) is the most frequent malignancy diagnosed in childhood. Because of the development of combined chemotherapy and support therapy, long-term survival rates have improved dramatically. However, some children still suffer from relapse of leukemia. Therefore, it is important to investigate the potential mechanism of leukemogenesis.The retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is a newly identified tumor suppressor gene (TSG). Although down-regulation and silence of RIZ1 mRNA expression is commonly detected in diverse human solid tumors, functions of RIZ1 gene in patients with hematologic malignancy is still unclear. Epigenetic alterations, such as DNA methylation, change structure of chromatin without involving the change in DNA sequences, resulting in silence of tumor suppressor gene expression, and contribute to carcinogenesis. Aberrant DNA methylation has become hot spot investigation on carcinogenesis. The methylation reaction is mediated by a class of enzymes termed DNA methyltransferases (DNMTs) that catalyze and maintain DNA methylation status. Increased expression of DNMTs is detected in several human tumors. DNMTs play a significant role in carcinogenesis.In the present study, we investigated RIZ1 mRNA expression and promotor CpG island methylation status of patients with acute leukemia at onset, remission and relapse stages, analyzed the relationship with clinical characteristics, and explored the possible mechanisms involved in leukemogenesis. We also studied the expression levels of DNMTs, as well as the connection between RIZ1 and DNMTs expression levels, and assessed the role of DNMTs in the process of acute leukemia. Part I Expression of RIZ1 Gene in Children with Acute LeukemiaObjective:The purpose of this study is to investigate the expression of RIZ1 gene in children with acute leukemia, and clinical values, to initially explore the role of RIZ1 gene in leukemogenesis.Methods:We examined the mRNA expression of RIZ1 gene in 156 patients with pediatric acute leukemia (92 ALL and 64 AML; 77 male and 79 female; mean age 6.43 years old), by using quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between RIZ1 expression and clinical characteristics has been analyzed. Bone marrow sample from 13 children with non-neoplastic hematologic diseases were collected and assigned to the control group.Results:Compared with control, expression levels were significantly decreased in 92.4% ALL and 96.9% AML (P=0.001 and P<0.001). At remission, onset, and relapse stages, expression levels were gradually reduced in patients with ALL. Lower expression of RIZ1 was detected in T-ALL than in B-ALL, but there was no statistic difference. Expression levels of were gradually decreased in SR, MR and HR groups (P<0.001). At remission, expression level was lower than onset, and higher than relapse stage in patients with AML. However, there was no significant difference between onset and relapse. The same trend in risk-based subgroups of AML patients was identified as the changes in ALL. Self-control study was used in 33 ALL and 22 AML, and found that expression significantly increased after remission (P=0.005 and P<0.001). At different stages, RIZ1 expression levels in AML children were lower than in ALL.Conclusion:RIZ1 expression in patients with ALL and AML is decreased. Down-regulated expression of RIZ1 gene may serve as an molecular marker of adverse prognosis in pediatric acute leukemia. Partâ…¡Aberrant Methylation of RIZ1 in Pediatric Acute LeukemiaObjective:To investigate the correlation between methylation status of promotor CpG islands and down regulated expression of RIZ1 gene. And explore the role of methylation in leukemogenesis.Methods:Methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing were performed to detect methylation status of RIZ1 promotor CpG islands in newly diagnosed ALL and AML (46 male and 50 female; mean age 6.43 years old). The correlation between DNA methylation and down regulated expression of RIZ1 gene, and effect on prognosis was also assessed. Nine children with non-neoplastic hematologic diseases were assigned to the control group.Results:Among 96 patients, DNA methylation of RIZ1 promotor CpG islands was identified in 27 children (28.1%), and methylation frequency was 23.4% in ALL and 37.5% in AML, while none of control showed methylation of RIZ1. At remission, onset, and relapse stages, methylation frequencies were gradually reduced in patients with ALL (P=0.006). There was no significant difference between T-ALL and B-ALL (P=0.565). Methylation frequencies were gradually increased in SR, MR and HR groups (P=0.003). There was no statistic difference in patients with AML at different stages (P=0.303). Each child classed into LR group was detected as unmethylated status. With elevated risk, frequencies in subgroups were increased gradually (P=0.002). Expression levels of RIZl gene were significantly reduced in methylated patients than in unmethylated (P<0.001).Conclusion:DNA methylation of RIZ1 gene is not such a frequent events, but plays an important role in decreased expression of RIZ1. Promotor methylation of RIZ1 is related to the process of pediatric acute leukemia, and may be an adverse prognostic factor of childhood leukemia. Partâ…¢Correlation Between DNA methyltransferases expression and RIZ1 Downregulation in Pediatric Acute LeukemiaObjective:To explore the expression of DNA methyltransferases in children with ALL and AML, and their contribution to RIZ1 expression and CpG islands methylation.Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression of DNMTs (including DNMT1, DNMT3a and DNMT3b) in 41 ALL and 29 AML patients (35 male/female; mean age 6.25 years old). Thirteen children with non-neoplastic hematologic diseases were collected as the control group.Results:Compared with control group,3.86-,4.44-, and 8.21-fold increase of DNMT1, DNMT3a, and DNMT3b, respectively, were detected in ALL patients, while 4.35-,4.57-, and 9.08-fold increase in AML. Expression level of DNMT1 was higher in HR group than SR (P=0.046), but DNMT3a and DNMT3b expression had no significant difference between groups in ALL. The same results were observed in AML patients (DNMT1, P=0.05). Expression levels of DNMT1, DNMT3a and DNMT3b in methylated ALL patients were higher than in unmethylated (DNMT1, P=0.015; DNMT3a, P=0.038; DNMT3b, P=0.003). DNMT1 and DNMT3b expression levels were higher in methylated AML patients than that in unmethylated patients (DNMT1, P=0.007; DNMT3b, P<0.001), but expression of DNMT3a had no significant difference between these children (P=0.131).Conclusion:Expression of DNMTs were significantly increased in patients with pediatric acute leukemia, and expression in methylated patients were higher than unmethylated. Up regulation of DNMTs may contribute to the promotor methylation of RIZ1 gene, and participate in leukemogenesis.
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