Effects of Bupleurum chinense on the diversity of intestinal flora in depressed mice / 中国中药杂志

Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.

Investigation of PRAM1 Expression Features and Their Clinical Significance in AML via Gene Expression Microarray Database / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the clinical phenotype and its prognostic value of PRAM1 in patients with acute myeloid leukemia(AML).</p><p><b>METHODS</b>Based on the gene expression microarray platform of 486 AML cases, the PRAM1 expression phenotypes were summarized in all of AML subtypes. The PRAM1 expression features were explored in every differentiation stage of hematocytes through normal human stem cell chips and bone marrow gene expression microarray. The clinical drugs which could up-regulate PRAM1 expression in AML cell lines should be found out.</p><p><b>RESULTS</b>The PRAM1 expression was the richest in the inv(16) AML and the lowest in the t(15;17)M3, almost the same in the other subtypes of AML. By the classification of molecular abnormalities, PRAM1 expression was more in the panel of CN-AML with CEBPAdm than the other two panels. Interestingly, high/low expression of PRAM1 could be re-classified in the CN-AML, and the EFS is statistically significant. It was proven again that PRAM1 is more expressed in the mature granulocytes. Finally, it was confirmed that decitabine and the chidamide could up-regulate PRAM1 expression in AML cell lines, and chidamide effect is better.</p><p><b>CONCLUSION</b>PRAM1 expression is the lowest in t(15;17) M3 and the highest in inv(16) AML. The high expression of PRAM1 is a sign for favorable prognosis in the CN-AML. PRAM1 is more expressed in mature granulocytes, chidamide can up-regulate PRAM1 expression in AML cell lines.</p>

Research Progress of Ras/Raf/MEK/ERK Signaling Pathways in Leukemia-Review / 中国实验血液学杂志

Ras/Raf/MEK/ERK signaling pathway plays an important role in the occurrence and development of leukemia. Using the inhibitors of key signaling components in the signaling pathway is new strategy for the treatment of leukemia. In recent years, the screening of these inhibitors and their in vitro and in vivo researches have become a hot spot in the field of treatment. In vivo and in vitro experiments and early clinical studies have shown that these inhibitors have good effect and application prospects in the treatment of leukemia. This review focuses on the recent advances of the role of Ras/Raf/MEK/ERK signaling pathway in the occurence, development and treatment of leukemia.

Clinical Features and Prognosis of t(8;21) AML Patients in China: A Multicenter Retrospective Study / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To summarize the clinical characteristics of peripheral blood, immune phenotypes, fusion genes and cytogenetics of patients with t(8;21) acute myeloid leukemia(AML) through the retrospective analysis of 586 patients with t(8;21) AML from 15 blood disease research centers in Northern area of China.</p><p><b>METHODS</b>The factors affecting prognosis of patients with t(8;21) AML were investigated by using univariate and multivariate COX regression.</p><p><b>RESULTS</b>The immune type of t(8;21) AML patients was mainly with HLA-DR, CD117, CD34, MPO, CD38, CD13and CD33(>95%), part of them with CD19and CD56; the most common accompanied mutation of t(8;21) AML patients was C-KIT mutation (37.8%); in addition to t(8;21) ectopic, the most common chromosomal abnormality was sex chromosome deletions (38.9%). The univariate analysis revealed a significant survival superiority of OS and PFS in t(8;21) AML patients of WBC≤3.5×10/L without C-KIT mutation, the newly diagnosed ones achieved HSCT(P<0.05), only survival superiority on OS in t(8;21) AML patients with extramedullary infiltration and CD19 positive; the results of multivariate analysis showed a significant survival superiority on OS and PFS in t(8;21) AML patients with WBC≤3.5×10/L(P<0.05).</p><p><b>CONCLUSION</b>The clinical features of t(8;21) AML patients in China are similar to those in other countries, WBC≤3.5×10/L is a good prognostic factor while the C-KIT mutation is a poor one in t(8;21) AML patients.</p>

Analysis of Clinical Characteristics in 10 Patietns with T Large Granular Lymphocytic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical manifestations and laboratory features of patients with T large granular lymphocytic leukemia (T-LGLL), so as to improve the understanding of this disease.</p><p><b>METHODS</b>The clinical data of 10 patients with T-LGLL in General Hospital of Chinese PLA from October 2015 to March 2010 were analyzed retrospectively.</p><p><b>RESULTS</b>Their median age at diagnosis was 51 years old. 9/10 (90%) patients showed symptoms of anemia, with a median Hb level of 82.5 g/L, 5/10 (50%) patients combined with autoimmune disorders and with a median Hb level of 77 g/L. 7/10 (70%) patients had splenomegaly, 2/10 (20%) patients had complex karyotype, 2/10 (20%) patients had gene mutations, the median age of 4 patients with complex karyotype and gene mutation was 49 years old, all of them suffered from splenomegaly. The immunophenotype of 6/10 patients was CD3+ CD4- CD8+ and that of 2/10 patients (20%) was CD3+ CD4- CD8-, that of another 2/10 (20%) was CD3+ CD4+ CD8-, the clinical features between different types of immunization were not statistically different.</p><p><b>CONCLUSION</b>T-LGLL patients often are old men, combined with anemia and splenomegaly, often associated with autoimmune diseases; the patients with complex karyotype and gene mutation are younger and they are more with hepatosplenomegaly; the guide role of different immunotypes for clinical strategy is no significant.</p>

Retrospective Analysis of the Impact of Treatment on Prognosis in 44 Adolescents and Young Adult Patients with Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the clinical characteristics and the impact of treatment on prognosis in 44 adolescents and young adult (AYA) patients with acute lymphoblastic leukemia.</p><p><b>METHODS</b>Clinical data of 44 AYA ALL patients admitted in our hospital from September 1997 to April 2014 were analyzed retrospectively and the impact of treatment on overall survival (OS) and event free survival (EFS) were investigated.</p><p><b>RESULTS</b>The median age of the patients at diagnosis was 23.7 (15-37) years and the male/female was 2.38:1. Out of them 88.6% of patients achieved complete remission (CR) after 1 course of induction chemotherapy, 35 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) and 6 patients received chemotherapy, 3 patients received autologous hematopoietic stem cell transplantation (auto-HSCT) as consolidation therapy in CR1. The expected 3-year OS and EFS rates of all the 44 patients were 64.3% and 61.7% respectively. The expected 5-year OS and EFS rates were 55.4% and 56.6% respectively. Allo-HSCT was not superior to chemotherapy and auto-HSCT in all the 44 patients (P = 0.308 for OS and P = 0.291 for EFS). In allo-HSCT group, the treatment related mortality was 22.9%, and the differences of OS and EFS in standard risk and poor risk AYA ALL patients were no significant (P = 0.775 for OS and P = 0.817 for EFS). However, compared with chemotherapy and auto-HSCT, allo-HSCT could significantly improve the OS and EFS in standard risk AYA ALL (P = 0.0296 for OS and P = 0.0359 for EFS).</p><p><b>CONCLUSION</b>Allo-HSCT as consolidation therapy may provide survival improvement for standard risk AYA ALL. However, further prospectively randomized clinical study is warranted to confirm whether allo-HSCT is an optimal treatment for AYA ALL, which is still controversial at present.</p>

Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.</p><p><b>METHODS</b>Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.</p><p><b>RESULTS</b>EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.</p><p><b>CONCLUSIONS</b>Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.</p>

A Retrospective Study of Ph Negative Adolescent and Young Adults with Acute B Lymphoblastic Leukemia in Two Centers / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the therapeutic efficacy of different consolidation therapies after induction remission on Ph negative adolescent and young adults with acute B lymphoblastic leukemia, and to explore the effect of different risk factors on prognosis.</p><p><b>METHODS</b>The treatment and efficacy of 80 Ph negative B-ALL in patients of 16-39 years old in the Hematology Department of 301(65 cases) and 309(15 cases) hospital from 1999 to 2016 are retrospectively analyzed. The patients received combined induction chemotherapy of 4 or 5 chemotherapeutic drugs (VDCLP/ VDLP/ DOLP/ IOLP). After remission patients received consolidation protocols of 3-5 cycls, and then received allo-HSCT or haploidentical HSCT. The median follow-up time was 29 (6-153) months.</p><p><b>RESULTS</b>HSCT was carried out after CR1. The 5-year OS and EFS of allo-HSCT group(n=29) was (73±16)% and (67±17)%, respectively, while those of haploidentical-HSCT group(n=20) were (53±22)% and (53±22)%, respectively, and those of pediatric-inspired protocols(n=31) was (63±17)% and (50±18)%, respectively. The difference between OS and EFS in 3 group was not statistically significant(P>0.05). The re-remission rate of recurrent patients was (50±23)%. On the one side, the cumulative incidence of TRM of pediatric-inspired protocol was better than that of HSCT (P<0.05). On the other side, the cummulative incidence of relapse (CIR) of pediatric-inspired protocol was poorer than that of HSCT, yet without significant difference (P>0.05). The median remission time of CR2 in patients was 14(2-36) months. Univariate and multivariate analysis were performed in 65 patients, and showed an abnormal result of CD13 or CD33 positive, CD22 negative, indicating a poor prognosis(P<0.05).</p><p><b>CONCLUSION</b>In the adolescent and young adult patients with PhB-ALL treated by pediatric-inspired protocols, the survival time is similar with that in allo-HSCT group. However, more prospective clinical studies of random control test(RCT) should be carried out.</p>

Advances in the Therapy of t(8;21) Acute Myeloid Leukemia / 中国实验血液学杂志

The t(8;21)(q22;q22) translocation is the most common chromosomal abnormalities in AML, and the chromosomal translocation forms AML1-ETO. The t(8;21) AML is a heterogeneity disease. It is unclear for how to treat the relapsed or refractory AML. Recently, the clinical trials and pathogenesis have made great progress. This article summarizes the current clinical trials and recruiting t(8;21) AML clinical trials and researches that related to treatment are as followed: epigenetics, JAK/STAT signaling, steroid, Chinese traditional medicine, and interferon. With the progress of pathogenesis researches, more and more treatments will translate into clinical trials, which can provide more optional choice for relapsed or refractory t(8;21) AML. In this article the AML1-ETO structure and t(8;21) AML pathogenesis, the clinical researches of t(8;21) AML treatment and basic researches of t(8;21) AML treatment are summarized.

Genomics-based Approach and Prognostic Stratification Significance of Gene Mutations in Intermediate-risk Acute Myeloid Leukemia / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>Intermediate-risk acute myeloid leukemia (IR-AML), which accounts for a substantial number of AML cases, is highly heterogeneous. We systematically summarize the latest research progress on the significance of gene mutations for prognostic stratification of IR-AML.</p><p><b>DATA SOURCES</b>We conducted a systemic search from the PubMed database up to October, 2014 using various search terms and their combinations including IR-AML, gene mutations, mutational analysis, prognosis, risk stratification, next generation sequencing (NGS).</p><p><b>STUDY SELECTION</b>Clinical or basic research articles on NGS and the prognosis of gene mutations in IR-AML were included.</p><p><b>RESULTS</b>The advent of the era of whole-genome sequencing has led to the discovery of an increasing number of molecular genetics aberrations that involved in leukemogenesis, and some of them have been used for prognostic risk stratification. Several studies have consistently identified that some gene mutations have prognostic relevance, however, there are still many controversies for some genes because of lacking sufficient evidence. In addition, tumor cells harbor hundreds of mutated genes and multiple mutations often coexist, therefore, single mutational analysis is not sufficient to make accurate prognostic predictions. The comprehensive analysis of multiple mutations based on sophisticated genomic technologies has raised increasing interest in recent years.</p><p><b>CONCLUSIONS</b>NGS represents a pioneering and helpful approach to prognostic risk stratification of IR-AML patients. Further large-scale studies for comprehensive molecular analysis are needed to provide guidance and a theoretical basis for IR-AML prognostic stratification and clinical management.</p>

Clinical Characteristics and Prognosis of 34 Patients with Mantle Cell Lymphoma: A Retrospective Analysis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the clinical characteristics and prognostic factors of 34 patients with mantle cell lymphoma (MCL).</p><p><b>METHODS</b>Clinical records of 34 MCL patients admitted from October 2007 to February 2015 in our hospital were analyzed retrospectively, the influcence of clinical characteristics, therapeutic protocol and biological indicators on overall survival (OS) was investigated.</p><p><b>RESULTS</b>The median age of the patients at diagnosis was 58.4 years, with a significant male predominance (3.25: 1), 58.8% of patients had bone marrow involvement, 29.4% had alimentary tract involement, and 79.4% were in Ann Arbor stage III-IV. The expected 3 and 5-year overall survival (OS) rates of all the 34 patients were 63.6% and 55.6% respectively. Rituximab in combination with chemotherapy was not significantly superior to chemotherapy alone in terms of OS in this study (68.4 months vs. 51.8 months, P = 0.979). In univariate analysis, absolute monocyte count (AMC) >0.375 × 10(9)/L and increased lactate dehydrogenase (LDH) level at diagnosis were associated with poor OS (P < 0.05).</p><p><b>CONCLUSION</b>Most patients were diagnosed at advanced stage. Rituximab plus chemotherapy can prolong OS time, but no statistically significant difference is observed, which maybe due to the fewer patients in this retrospective study. The high level of AMC and LDH at diagnosis are poor prognostic factors.</p>

Markers for Predicting Response to Hypomethylating Agents in MDS and AML Patients / 中国实验血液学杂志

In many countries the hypomethylating agents (HMAs) are the major compounds to treat patients suffering from myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) with low bone marrow blast counts. The elderly AML patients with poor performance status and comorbidity also benefit from HMAs. Patients who resist to HMA have a poorer outcome as compared to sensitive patients. Therefore, the reliable markers for predicting response to HMAs are urgeatly needed in clinic. In this review, the biomarkers for predicting response of patients with MDS or AML to HMAs are summarized.

Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The diagnosis of myelodysplastic syndrome (MDS), especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA).</p><p><b>METHODS</b>The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA.</p><p><b>RESULTS</b>The MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0-3.79]) than the AA group (P < 0.05). Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]).</p><p><b>CONCLUSIONS</b>These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.</p>

Construction of SIRT1 promoter expression vector and its activity analysis as well as influence of AML1-ETO on transcriptional regulation of SIRT1 gene / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to analyze the expression and regulation mechanism of SIRT1 in AML1-ETO positive leukemia to find the core promoter.</p><p><b>METHODS</b>The real-time RT-PCR was used to detect the expression of SIRT1 in AML1-ETO positive leukemia cell line and clinical samples of leukemia patients, a SIRT1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the 293T cell line. A series of possible core promoter fragments of the SIRT1 5'-untranslated region were amplified by PCR, the PCR products were cloned into XhoI/HindIII-digested pGL3-Basic reporter vector, the poly-cationic compound SuperFect reporter vector complexes were transfected into 293T cells.The dual-luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity.</p><p><b>RESULTS</b>The six kinds of promoter fragment of SIRT1 gene were successfully constructed and cloned into the pGL3-Basic reporter vector, which was authenticated by XhoI/HindIII co-digestion and DNA sequencing. The luciferase activity of the promoter construct was significantly higher than that of the pGL3-Basic promoter in 293T cells. The luciferase report gene assay was also used to detect the regulation of AML1-ETO on the transcription activity of SIRT1 promoter. The results showed that the expression level of SIRT1 increased with the increase mens of AML1-ETO, the promoter of SIRT1 could be bound by AML1-ETO.</p><p><b>CONCLUSION</b>The SIRT1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in 293T cells. The SIRT1 core promoter possesses higher activity in 293T cells and can promote significantly expression of luciferase reporter gene in 293T cells. The transcription regulation of AML1-ETO on SIRT1 is carried out via promoting its promoter activity.</p>

Effect of autologous peripheral blood stem cell transplantation in 13 patients with AML1/ETO (+) acute myeloid leukemia / 中国实验血液学杂志

This study was purposed to analyse the clinical efficacy of autologous peripheral blood stem cell transplantation (APBSCT) in 13 Patients with AML1/ETO (+) acute myeloid leukemia, and to evaluate the role of quantitative detecting the AML1/ETO gene in treatment of AML patients. A total of 13 patients with AML1/ETO (+) acute myeloid leukemia treated with APBSCT from August 2007 to November 2012 were retrospectively analyzed. The median follow-up time was 26 (7.8-75.8)months. Kaplan-Meier analysis was used to calculate the overall survival (OS), leukemia-free survival (LFS) and cumulative relapse rate (RR). Log rank method was used to perform univariate analysis. The results showed that the 3 year-OS, LFS, and RR were (70.5 ± 15.3)%, (51.3 ± 16.7)%, 48.7%, respectively. The AML1/ETO expression level in 4 cases out of 5 relapsed patients was quantified during and after therapy, and the result showed that AML1/ETO expression level significantly increased before morphological relapse. In univariate analysis, there was no statistic significance in terms of age, sex, count of white blood cells at diagnosis, interval from diagnosis to transplantation, count of MNC for infusion. It is concluded that APBSCT has good therapeutic effect on AML1/ETO (+) AML, and regular quantitative monitoring of AML1/ETO expression level can predict early recurrence. Allogeneic hematopoietic stem cell transplantation after relapse may contribute to obtain opportunity to achieve the long-term survival for intermediate and high risk patients.

Demethylating agent decitabine induces autologous cancer testis antigen specific cytotoxic T lymphocytes in vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cancer testis antigens (CTAs) are a novel group of tumor associated antigens. Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism, thus enhance the immunogenicity of leukemia cells. However, few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo, and if so, whether this effect contributes to disease control. In this study, we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.</p><p><b>METHODS</b>Several mouse CTAs were screened by RT-PCR. CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry. The activity of specific CTLs was measured by real time RT-PCR.</p><p><b>RESULTS</b>We firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs. Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment. Finally, we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.</p><p><b>CONCLUSIONS</b>Our study showed the autologous immune response induced by decitabine in vivo. And more importantly, we firstly proved that this response may contribute to disease control. We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine, and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.</p>

Poliovirus receptor on surface of acute B lymphoid leukemia cells modulated by epigenetic mechanism / 中国实验血液学杂志

The aim of this study was to identify if the expression of poliovirus receptor (PVR) on the surface of acute B lymphoid leukemia (B-ALL) cells RS4:11 and SUP-B15 is modulated by epigenetic mechanism. B-ALL cell lines RS4:11 and SUP-B15 were treated with demethylation agent. Bisulfite PCR was performed to detect percentage change of the methylated CpG islands in the promoter region of PVR. In the meantime, the expression levels of PVR at the translation and transcription levels were detected by flow cytometry and RT-PCR respectively. The B-ALL cell lines were also treated with histone deacetylase (HDAC) inhibitor. The expression level of the gene mRNA and protein was detected too. The results indicated that after treated with 5-azacytidine, the hypermethylated status of PVR promoter region was partly reversed, and the expression of PVR at both mRNA and protein levels was restored in the meanwhile. HDAC inhibitor suberoylanilide hydroxamic acid could also increase the PVR expression. But there was no synergistic function between hypermethylation and HDAC as for repressing PVR transcription in B-ALL cell lines. It is concluded that the expression of PVR in B-ALL cells is modulated by epigenetic mechanisms. Treatment with corresponding inhibitors can partly restore the gene's expression in both mRNA and protein levels.

Significance of methylation status of zo-1 gene in differential diagnosis for myelodysplastic syndrome / 中国实验血液学杂志

It is hard to discriminate myelodysplastic syndrome(MDS) from many benign hematological diseases. To identify the methylation status of zo-1 gene in MDS, the methylation specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to detect the MDS cell line MUTZ-1, bone marrow of a healthy donor and an aplastic anemia patient. MS-PCR was also employed to detect the bone marrow of 72 patients with benign hematological diseases, 35 MDS-RA patients, and 20 MDS-like patients. The results showed that MDS cell line MUTZ-1 displayed complete methylation of zo-1 promoter without mRNA expression. Inversely, a patient with benign hematological disease and a donor with normal bone marrow showed complete unmethylation of this gene with unaffected mRNA expression. No zo-1 promoter methylation was detected in patients with benign hematological diseases, while aberrant hypermethylation of zo-1 gene promoter were found in 48.6% (18/37) of MDS-RA patients. The positive rate of zo-1 methylation in MDS-RA patients was higher than that in patients with benign hematological diseases (p < 0.05). Seven suspected MDS patients manifested hypermethylation status of zo-1 gene (7/20), 2 were followed up for 1 year and transformed into MDS. It is concluded that relatively high hypermethylation rate of zo-1 promoter is observed in MDS-RA, and no methylation in patients with benign hematological diseases. Therefore, zo-1 gene hypermethylation may be served as a useful epigenetic marker in the differential diagnosis for MDS.

Expression level of miRNA-663 in different leukemic cell lines and its biological function / 中国实验血液学杂志

The aim of the research was to find the up-regulated microRNA (miRNA) in K562 cells treated by 5-aza, to detect the miRNA expression in healthy people, CML patients and leukemia cell lines and to investigate the influence of miRNA on K562 cell proliferation. Up-regulated miRNA in K562 cells after 5-aza treatment was screened by microarray. The up-regulated miR-638, miR-663 and miR-92b with CpG islands in upstream region were screened by microarray in combination with bioinformatics. The miRNA mentioned above was further ascertained by SYBR-green real-time PCR. Up-regulated miR-663 was confirmed by real-time PCR. Expression level of miR-663 was detected in K562, U937 and Kasumi cell lines, and white blood cells from bone marrow of normal donor and from peripheral blood of newly diagnosed CML patient. Methylation-specific PCR (MSP) was applied to analyze the methylated status of miR-663 CpG island in K562 cells. Proliferation of K562 cells was observed after miR-663 was over expressed by transient transfection. The results showed that the expression level of miR-663 in K562 cells was up-regulated after 5-aza treatment, and the expressions of miR-663 were lower in K562, U937, Kasumi cell lines and newly diagnosed patients, compared with healthy people. The CpG island of miR-663 was methylated in K562 cell line according to detection result of MSP. The proliferation of K562 cell could be suppressed by over-expression of miR-663 in vitro. It is concluded that miR-663 CpG island is methylated in K562 cell line. The miR-663 is down-regulated in K562, U937, Kasumi cell lines and CML patients, compared with healthy people. miRNA-663 in K562 cells is up-regulated after 5-aza treatment. Over-expression of miR-663 can suppress the proliferation of K562 cells, which suggests that miR-663 may possesses suppressive effect for leukaemia.

Methylation status of id4 gene promoter in patients with chronic myeloid leukemia / 中国实验血液学杂志

This study was purposed to investigate the methylation status of id4 gene promoter in patients with chronic myeloid leukemia (CML) and explore the relationship between methylation of the id4 gene and progress of CML. The methylation status of id4 gene in 48 chronic myeloid leukemia patients and 10 healthy individuals was detected by using methylation-specific polymerase chain reaction (MS-PCR). The results showed that id4 gene was unmethylated in bone marrow samples from both healthy individuals and CML patients in chronic phase (CP). The rate of id4 gene methylation in both CML patients in accelerated phase (AP) and blast crisis (BC) was 66%, and was higher than those of CML patients in CP phase. There was significant difference between them (p < 0.05). In one CML patient who received a serial observations, the status of id4 was unmethylated in CP, but it was methylated in AP and BC phase. It is concluded that the id4 gene in CML patients is unmethylated in CP, while it is methylated in AP or BC. The detection of id4 gene methylation status may be useful for monitoring disease advance in CML and may be used as a marker of disease progression in CML.