The Role Of ZHX2 In Adults B-ALL And Its Mechanism, And Aberrant Circulating Th Subsets In Patients With Lymphoma

Section IThe role of ZHX2 in adults with B-cell acute lymphoblastic leukemia and its mechanismBackground:Acute lymphoblastic leukemia (ALL) is a clonal progressive malignant disease derived from B or T cell progenitors. These malignant cells suppress the normal hematopoiesis in bone marrow and invade extramedullary tissue. So far, the reason of B-ALL is still undefined. Although therapeutic strategies cure more than 80% of children with acute lymphoblastic leukemia, survival rate for adult patients remains below 40%. Novel approaches are needed to increase efficacy and prolong survival for adult B-ALL patients.ZHX2, which is a new member of ZHXs family, has been described as a transcriptional repressor and implicated in several human diseases. In multiple myeloma, increased expression of ZHX2 is associated with favourable prognostic indicators and more prolonged survival. In Hodgkin’s lymphoma, knockdown of ZHX2 in HL-derived cell line (L-1236) has showed inhibition of genes regulating apoptosis. By gene expression profiling ZHX2 gene may play an important role in human B cell development. These research results lead us to investigate the biological function of ZHX2 in adult B-ALL.Objective:In our current study, we investigated the role of ZHX2 played in adult B-ALL disease. Our data showed that ZHX2 mRNA levels were decreased in bone marrow samples from adult B-ALL patients and high ZHX2 was associated with better disease progression and longer overall survival of patients. We also revealed that up-regulation of ZHX2 could inhibit B-ALL cells proliferation and enhance cell sensitivity to chemotherapy. Moreover, we also tried to find out the mechanism of these results.Methods:1. Patient samples:Bone marrow samples from 37 newly diagnosed adult acute B lymphoblastic leukemia patients and 30 normal donors were obtained after informed consent at the Qilu Hospital, Shandong University from January 2012 to September 2014. Mononuclear cells (MNCs) were isolated from bone marrow aspirates by Ficoll-Hypaque density-gradient centrifugation and stored at -80℃.2. Real-time RT-PCR:Total RNA was obtained from cells and patient samples using TRIzol reagent. Real-time RT-PCR was performed with SYBR Green PCR Master Mix in a 10 μl reaction volume and normalized by GAPDH.3. Western blotting:Cell extracts by cells lysed in protein extraction buffer mixed with protease and phosphatase inhibitors. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) with exposure to autoradiography film.4. Cell proliferation assay:Cell Counting Kit-8 assay was conducted to assess cell proliferation. The absorbance was measured at 450 nm (620 nm as a reference absorbance). Each experiment was repeated at least 3 times.5. Cell cycle assay:For cell cycle analysis, cells were collected and stained with propidium iodide and assayed using a Beckman Gallios Flow Cytometer. Each experiment was repeated at least 3 times.6. Statistical Analysis:GraphPad prism 5.0 was used for data analysis. The Student t-test, Mann-Whitney U test or Kruskal-Wallis test was applied to determine significant differences between groups. Two-way ANOVA was applied to determine significant differences between different treatments, in different cell cohorts or at different time points. Correlation analysis was determined by Spearman’s test. The statistical correlation between the clinical characteristics and ZHX2 expression level in patients was analyzed by the Chi-square test. All survival analyses refer to overall survival times (OS), in which death from any cause represents an event. Median OS within each subgroup were estimated from Kaplan-Meier curves and survival differences were analyzed using the log-rank test. In these analyses, two-sided P-values <0.05 were considered to be statistically significant.Results:1. By using real-time RT-PCR analysis, the result showed that ZHX2 mRNA expression was significantly reduced in newly diagnosed adult B-ALL patients than normal controls2. The mRNA expression of ZHX2 in newly diagnosed adult B-ALL patients was related with their clinical characteristics:(1) There was no significant difference about ZHX2 mRNA levels between Ph positive and Ph negative groups.(2) Higher ZHX2 mRNA levels were associated with the younger age (15-39 years) which was considered as a low-risk feature in adult B-ALL.(3) Sequentially, higher ZHX2 expression showed a correlation with low-risk features in Ph positive group.3. ZHX2 played benefit roles in adult B-ALL patients’outcome and progression:(1) Higher ZHX2 expression had significantly better overall survival compared to the remaining B-ALL patients.(2) There was a trend that the duration of CR was longer in patients with higher ZHX2 levels compared with lower ZHX2 levels.(3) In Ph+ B-ALL, the patients with higher ZHX2 expression had significantly better overall survival compared to those with lower ZHX2 expression.4. Overexpression of ZHX2 could inhibit the proliferation of B-ALL cells.5. Overexpression of ZHX2 in Nalm-6 cells increased the percentage of G0/G1 cells, without effect on percentage of G2/M or S phase cells.6. Higher ZHX2 expression could enhance chemotherapeutic sensitivity in B-ALL cells.7. ZHX2 overexpression enhanced Notchl-activation and increased p27 level in B-ALL cells.Conclusion:1. The mRNA expression of ZHX2 was decreased in newly diagnosed adult B-ALL patients.2. The mRNA expression of ZHX2 was related with their clinical characteristics, and ZHX2 played benefit roles in adult B-ALL patients’outcome and progression.3. Overexpression of ZHX2 could inhibit the proliferation of B-ALL cells and enhance chemotherapeutic sensitivity in B-ALL cells, indicating that ZHX2 could be considered as a tumor suppressor in B-ALL.4. Further researches should be done to make ZHX2 as an effective therapeutic pathway against adult B-ALL.Section IIAberrant circulating Th subsets in patients with lymphomaBackground:Lymphoma is a heterogeneous group of malignancies originating in lymphatic hematopoietic tissue. According to the histologic classification, it can be classified into non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL). According to different types of lymphoid cells, it also can be classified into B-cell lymphoma and T-cell lymphoma. Although chemotherapy has been used as the main therapy for patients with lymphoma, resistance of tumor cells to chemotherapy often results in the failure of treatment. Therefore, new targets and methods are required to improve the outcome of the patients.In recent decades, may studies have demonstrated that T helper (Th) cells participate in the pathogenesis and development of autoimmunity and inflammatory disease such as experimental allergic encephalomyelitis, autoimmune arthritis, multiple sclerosis and psoriasis. However, the specific role of Thl7 and Th22 cells in tumor are still certain and debatable. Up to now, no previous study has shown data about the roles of Th17, Th22 subset and their related cytokines in the pathogenesis and development of lymphoma, especially B-cell non-Hodgkin’s lymphoma.Objective:In our current study, we investigated the frequency of Th1 (CD+IFN-y+), Th17 (CD4+IL-17+), Th22 (CD4+IFN-y"IL-17"IL-22+) cells in the peripheral blood, concentrations of plasma IL-17AA, IL-17FF, IL-17AF, IL-22 and IL-6 in patients with B-NHL. We also evaluated the relevance between Thl, Th17, Th22 cells and clinical characteristics or disease’s development in this study.Methods:1. Patient samples:A total of 57 patients with lymphoma (21 females and 36 males, age range 18-79 years old, median 59 years old) were collected in this study. Collection took place from January 2013 to December 2013 at the Department of Hematology, Qilu Hospital, Jinan, China. All cases were consistent with lymphoma diagnostic criteria. Thirty-nine healthy controls (21 females and 18 males, age range 20-50 years old, median 32 years old) were included. Study was approved by the Medical Ethical Committee of Qilu Hospital, Shandong University and all participants gave written informed consent.2. Flow Cytometric Analysis:Briefly, heparinized peripheral blood was incubated with the presence of PMA, ionomycin and monensin. After incubation, the cells were stained with anti-CD4, anti-IFN-y, anti-IL-17 and anti-IL-22 antibodies. Stained cells were analyzed by flow cytometric analysis using a FACS Calibur cytometer equipped with CellQuest software.3. Enzyme-linked immunosorbent assay (ELISA):Peripheral blood was collected into heparin-anticoagulant vacutainer tubes. All plasma specimens were obtained from all subjects by centrifugation and stored at -80℃ for determination of cytokines. The concentrations of IL-17AA, IL-17FF, IL-17AF, IL-22 and IL-6 in each group were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer’s recommendations. The concentrations were calculated from a standard curve according to the manufacturer’s protocol. Sensitive concentration of IL-17AA, IL-17FF, IL-17AF, IL-22 and IL-6 ELISA kit is 0.5pg/ml,15.5pg/ml,8.8pg/ml, 5pg/ml and 0.92pg/ml respectively.4. Statistical Analysis:The results were expressed as median range. Student t-test, Wilcoxon signed rank test, Mann-Whitney U test or Kruskal-Wallis test was applied to determine significant differences between groups. All tests were performed by GraphPad Prism 5.0 system. In these analyses, two-sided P-values<0.05 were considered to be statistically significant.Results:1. Aberrant circulating Th subsets in patients with lymphomas:(1) Increased Thl cells in the newly diagnosed non-Hodgkin’s lymphoma rather than in Hodgkin’s lymphoma(2) Decreased Th17 cells in the newly diagnosed B-cell non-Hodgkin’s lymphoma neither T-cell non-Hodgkin’s lymphoma nor Hodgkin’s lymphoma.(3) Circulating Th22 cells were elevated in newly diagnosed non-Hodgkin’s lymphoma rather than Hodgkin’s lymphoma.2. The concentrations of Th17 and Th22 related cytokines in newly diagnosed B-NHL:(1) Decreased IL-17AF with undetectable IL-17FF and non-changed IL-17AA in B-cell non-Hodgkin’s lymphoma.(2) Plasma IL-22 levels were up-regulated in newly diagnosed B-NHL patients, accompanied with elevated IL-6.3. Th22 cells showed positive correlations with Th17 cells in newly diagnosed B-NHL patients.4. B-NHL patients with older age (>60 y) exhibited profoundly increased frequency of circulating Th22 cells compared to patients with younger age (≤60y).5. The correlation of circulating Th subsets with the development of B-NHL patients:(1) The percentages of circulating Thl cells were statistically decreased and normalized in treated patients after one or two cycles of chemotherapy than those before treatment. Additionally, the percentages of Thl cells in relapsed patients were similar to those in newly diagnosed patients but still significantly higher than normal controls.(2) The percentages of circulating Th17 cells were statistically increased and normalized in treated patients after one or two cycles of chemotherapy than those before treatment. Interestingly, compared with untreated patients and even healthy individuals respectively, a significant increase of peripheral Th17 was seen in relapsed patients(3) The frequencies of Th22 cells in treated patients were significantly lower than those before any chemotherapy. We also found normalized circulating Th22 cells in these treated patients compared with normal individuals. Meanwhile, Th22 cells were remarkably increased in relapsed patients compared with in newly diagnosed patients and normal controls.Conclusion:1. The frequencies of circulating Th1, Th17 and Th22 cells were abnormal in peripheral blood from newly diagnosed patients with lymphomas, especially B-NHL.2. Th1, Th17 and Th22 cells were normalized after chemotherapeutic treatment.3. Th1, Th17 and Th22 cells may be associated with the response of patients to treatment and with different stages of disease in B-NHL.4. Further researches should be done to make sure the detail roles of Th subsets in B-NHL and try to make Th subsets as an effective therapeutic pathway against lymphoma.