Expression Of PTEN Gene In Chronic Lymphocytic Leukemia And Its Regulation

Part ⅠLow expression level of PTEN gene predicts poor prognosis in chronic lymphocytic leukemiaObjectives:Phosphatase and tension homolog deleted on chromosome ten (PTEN) is one of the best-studied tumor suppressor genes, which play a vital role in cell growth, development and apoptosis. In preliminary experiments, we found low level expression of PTEN mRNA (Pm) in chronic lymphocytic leukemia (CLL). The aim of this study is to investigate Pm expression level in a large of patients with CLL and explore its clinical significance.Methods:One hundred and three previously untreated CLL patients or patients who had not been treated for six months or more and20healthy controls were enrolled in the present study. Quantitative polymerase chain reaction (qPCR) was used to detect Pm expression. The p53mutations 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). Flow cytometry (FCM) was used to detect the expression of CD38and ZAP-70. The relationship of the quantification of Pm and the above prognostic factors were analysed.Results:Of the103patients and20controls, the-relative expression level of Pm spanned from0to3.1167(median was0.0237) and from0.0587to1.5911(median was0.3302), respectively. PTEN expression level was obviously lower in CLL samples (P<0.001). The ratio of PTEN expression was significantly decreased at Binet B+C (average expression level was0.0173) compared with Binet A (average expression level was0.0407), and high correlation was found between PTEN expression and Binet stage (P<0.001). The expression of PTEN was associated negatively with lactate dehydrogenase (LDH)(P=0.019), β2-microglobulin (β2-MG)(P=0.036), p53aberrations (P=0.005), cytogenetic abnormality (P=0.016) and ZAP-70(P=0.008). The other factors including IGHV and CD38exhibited no significant difference. In multivariate analysis, only advanced Binet stage (P=0.027) and p53aberration (P=0.007) were associated with low PTEN expression level. There was significant difference between the lower and higher groups (P=0.040) in time to first treatment (TTFT) by the Log-rank test statistics. The low group had a significantly reduced TTFT compared to the high expression group. According to the Cox multivariate survival analysis, Binet stage (P<0.001) was the only independent prognostic factor for TTFT.Conclusions:Low expression level of PTEN predicts poor prognosis in CLL. The low expression PTEN gene probably plays a considerable role in the prognosis of CLL. Part Ⅱ Study on the relationship between single nucleotide polymorphisms of PTEN and low expression of PTEN in chronic lymphocytic leukemiaObjectives:In preliminary experiments, we found the expression level of phosphatase and tension homolog deleted on chromosome ten (PTEN) was low in the chronic lymphocytic leukemia (CLL) patients. Mutations or deletions of PTEN gene have been found in various kinds of human tumors, which probably promote the occurrence and development of diseases. To study the mechanism of low expression level of PTEN, we explored the correlation between the single nucleotide polymorphism (SNP) of PTEN and its low expression in CLL patients.Methods:One hundred and fifty-four previously untreated CLL patients or patients who had not been treated for six months or more were collected. We sequenced exons5-9of PTEN gene in patients with CLL and200cases of healthy controls following polymerase chain reaction (PCR). The p53mutations and immunoglobulin variable heavy chain (IGHV) mutation status were also detected by PCR and direct sequencing. Cytogenetic abnormalities of patients were detected by interphase fluorescence in situ hybridization (FISH) analysis. Flow cytometric (FCM) analysis of CD38and ZAP-70were performed. The correlation between PTEN mutation and CLL prognostic factors were analysed.Results:None of the SNP site or mutation was detected in the coding sequences of exons5-9of PTEN gene. There was an SNP at intron8c.1026+32(rs555895) of PTEN gene in CLL and healthy controls. The genotype frequencies of T/T, T/G and G/G were24.0%,52.6%and23.4%in CLL patients, which were similar to those observed in the controls (17.0%,58.5%and24.5%, P=0.255). Compared with the controls (46.3%), there was no significant difference between T/T and (T/G+G/G) genotype in the CLL patients (P=0.102). There was also no statistical significance between the genotype frequencies of PTEN and prognostic factors of CLL patients.Conclusions:No PTEN gene mutation is identified in the CLL patients. PTEN gene mutation is not associated with the low expression in Chinese CLL patients. Part ⅢStudy on the correlation between methylation condition of PTEN promoter and low expression of PTEN in chronic lymphocytic leukemiaObjectives:Epigenetics produces lasting changes in gene expression without altering DNA sequence. Low expression level of phosphatase and tension homolog deleted on chromosome ten (PTEN) might be affected by epigenetics, such as DNA promoter methylation and microRNA (miRNA) interference. The purpose of this study is to investigate the relationship between the PTEN gene promoter methylation and its low expression.Methods:Seventy-five previously untreated CLL patients and25matched controls were collected. DNA samples were extracted from peripheral blood or the isolated lymphocytes. Nested methylation specific polymerase chain reaction (nMSP) using primers specific to sequences corresponding to either methylated or unmethylated DNA sequences was performed. Methylation results were validated by gene sequencing. EpiScope(?)Methylated HeLa gDNA was used as a positive control for methylated allele. The p53mutations and immunoglobulin variable heavy chain (IGHV) mutation status were analysed by polymerase chain reaction (PCR) and direct sequencing. Cytogenetic abnormalities of the CLL patients were detected by interphase fluorescence in situ hybridization (FISH) method. CD38and ZAP-70were detected by flow cytometry (FCM). The correlation between PTEN methylation and prognostic factors of the CLL patients was analysed.Results:Methylation of PTEN promoter was found in one (1.33%) of the75CLL patients and none of the controls, but PTEN methylation was detected in positive control of EpiScope(?)Methylated HeLa gDNA. These results were identified by directly sequenced. Those who were unmethylated in PTEN gene reveal a single nucleotide substitution of C→T, but the others had no change on single nucleotide sequence. The patient of altered DNA methylation showed poor clinical features of positive ZAP-70and deletion of the gene ataxia telangiectasia mutated (ATM).Conclusions:Methylation of PTEN promoter occurs rarely in the CLL patients, there is no association between the promoter methylation of PTEN gene and its low expression. Part ⅣStudy on the relationship between microRNA and low expression level of PTEN in chronic lymphocytic leukemia Objectives:As a form of epigenetics performance, microRNA (miRNA) interference plays an important role in regulating gene expression. The mature miRNA is incorporated into an RNA-induced silencing complex that binds to messenger RNA (mRNA) of a target gene, which causing oncogene activation or tumor suppressor gene activation. Patients with chronic lymphocytic leukemia (CLL) present a low expression level of anti-oncogene phosphatase and tension homolog deleted on chromosome ten (PTEN), which could be silenced by miRNA interference. The aim of this study is to determine the effect of miRNA interference on PTEN gene.Methods:Bioinformatics miRNA databases were used to identify PTEN-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 pEZX vector containing the complete3’untranslated regions (UTR) region of PTEN cloned in the3’UTR region of dual luciferase gene. For dual-luciferase assay,50nM,100nM,200nM and400nM of the miRNAs of interest or negative control (NC) were transfected in the293T cell line together with pEZX vector using lipofectamine2000, and luminescence was read after24h. Further validation of the miRNA target was performed by Western blot (WB) and reverse transcription polymerase chain reaction (PCR) in primary CLL cultured cells. Annexin/PI staining flow cytometry (FCM) observed the percentage of cell apoptosis. The p53mutations and immunoglobulin variable heavy chain (IGHV) mutation status were analysed by PCR and direct sequencing. Cytogenetic abnormalities of the CLL patients were detected by interphase fluorescence in situ hybridization (FISH) method. CD38and ZAP-70were analyzed by FCM.Results:The bioinformatics analysis identified six microRNAs as possible regulators of PTEN (hsa-miR-19a, hsa-miR-21, hsa-miR-26a, hsa-miR-26b, hsa-miR-214and hsa-miR-1297). The dual-luciferase assay confirmed that the mimics of hsa-miR-21, hsa-miR-26a and hsa-miR-214also suppressed the activity of PTEN. The analysis of these miRNAs showed that hsa-miR-21, hsa-miR-26a and hsa-miR-214were significantly downregulated after transfection with those miRNA inhibitors (P=0.014, P=0.040and P=0.001, respectively) in comparison with negative controls. Compared with controls, the apoptosis rates of the CLL cells were significantly increased after transfection with hsa-miR-26a (P<0.001) and hsa-miR-214(P=0.001) inhibitors. These findings were confirmed by PCR and WB analysis of PTEN levels after transfection with these two miRNA inhibitors.Conclusions:The low expression of PTEN is likely to correlate with aberrant hsa-miR-26a and hsa-miR-214. Inhibition of those two miRNAs may contribute to CLL cells apoptosis by increasing PTEN activity, which provides a novel therapeutic strategy in CLL.