The Effect Of Notch, VEGF, Ang Pathway And MicroRNA On Prognosis And Drug Resistanace In Myeloid Leukemia

Section I The impact of Notchl/D114, VEGF/VEGFR and Ang/Tie2pathway on pathogenesis and prognosis in acute myeloid leukemiaBackgroundAcute myeloid leukemia (AML) was a group of heterogeneous disease, and the pathogenesis of the disease involved in some genetic changes and multi-step processes. Although cytogenetic analysis provided a very powerful approach to discriminate prognosis of AML, there were some limitations of conventional cytogenetics. And the importance of molecular markers had been recognized in the prognosis of AML. Angiogenesis, the process of new blood vessel formation from an existing network of vasculature, played a pivotal role in the development and metastasis of tumor. Emerging evidence underscored the potential role of bone marrow (BM) angiogenesis in the progression, resistance and prognosis of AML Recent research indicated the key roles of Notch/D114, VEGF/VEGFR and Ang/Tie2in occurrence, development and angiogenesis of solid tumors. From our previous study, aberrant expressions of Notch1/D114and VEGF have been found in peripheral blood of AML patients. The role of Ang/Tie2and its relationships with Notchl/D114and VEGF/VEGFR in modulating angiogenesis were far from reached. And the effect of Notch1/D114, VEGF/VEGFR and Ang/Tie2on the prognosis of AML needed to be further research.ObjectiveTo investigate the expression and relationships of eight angiogenesis-related genes (D114, Notch1, VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2and Tie2) in BM mononuclear cells of adult patients with newly diagnosed AML, to explore the prognostic impact of the factors and clinical features in AML, and to provide the basis for pathogenesis and individual therapy with AML.Methods1. Sixty adult patients with untreated AML and40normal adult controls were enrolled in this study. The patients were devided into different subgroups according to age, sex, FAB subtype and karyotype. Clinical information for the patients were collected and the median follow-up duration was25months.2. BM mononuclear cells from AML patients and controls were separated by Ficoll-Hypaque density gradient centrifugation.3. Real time RT-PCR was used to detect the expression of angiogenesis-related genes The total RNA was isolated by Trizol and cDNA was prepared using the M-MLV Reverse Transcriptase. We established real-time quantitative RT-PCR (real-time RT-PCR) methods to detect the expression of angiogenesis-related factors (D114, Notch1,VEGF, VKGFR-1, VEGFR-2,Ang-1, Ang-2and Tie2) in BM mononuclear cells of patients with AML and GAPDH was detected as internal control.4. Lysis buffer solubilized cells and SDS-PAGE fractionated total proteins and transferred onto nitrocellulose membrane.Western blot method was used to detect Notch1and D114protein levels.5. All statistical analyses were performed with the SPSS13.0. Mann-Whitney U test, Analysis of variance and Kruskall-Wallis test were used to analyze the difference in the expression of angiogenic-factors between AML and control groups. Spearman’s test was used for the correlation between the individual expression of the genes studied, and the relationships of gene expression with clinical features. Kaplan-Meier method was applied to plot survival curves, and log-rank test was used to compare survival between groups. Univariate Cox proportional hazards regression models were used to evaluate the predictive effect of each factor alone on survival. Factors statistically significant in the univariate models at a5%level were included in the multivariate models. P<0.05were considered statistically significant.Results1. The mRNA expression of Notch1, D114, VEGF, VEGFR-2and Ang-2were significantly elevated in newly diagnosed AML patients compared with controls (P<0.05), while median levels of VEGFR-1, Ang-1and Tie2were similar between the two groups (P>0.05).2. The result of western blot analysis showed that the protein expression of Notch1and D114were up-regulated in untreated AML patients (P<0.001)3. The median VEGFR-2/VEGFR-1expression ratio was3.61-fold higher in AML patients than in controls (P=0.04). The median Ang-2/Ang-1expression ratio in AML patients was higher than controls (P=0.015).4. Significantly positive correlations were observed between Notch1and D1140=0.326; P=0.013), VEGF and VEGFR-20=0.354;P=0.007), Ang-1and Tie20=0.463; P=0.001). D114was closely associated with VEGF0=0.663; F=0.001), and Notchl correlated well with Ang-20=0.324; P=0.012).5. Correlation of angiogenic-factors expression with clinical features No significant differences were observed in the sex and FAB subtypes. The lever of Tie2was higher in AML patients under45years old than those over45years old (P=0.03), and the levels of D114and VEGF were significantly different among the karyotype subgroups (P=0.001). Significantly positive correlations of the percentage of leukemic blast infiltration were observed with the individual expression of Notchl0=0-428, P=0.001), D1140=0.288, P=0.026), VEGF0=0.428, P=0.001) and Ang-20=0.474, P<0.001).6. Univariate analysis of factors associated with OS revealed a significantly shorter survival in the patients with unfavorable karyotype, higher Notch1expression, higher D114expression, higher VEGF expression or higher Ang-2expression. Cox proportional hazards multivariate analysis of the univariate predictors identified karyotype and gene expression of Notch1, D114, VEGF and Ang-2as prognostic factors for OS (P<0.05).7. Kaplan-Meier survival curves showed that D114expression has a significant impact on OS in patients with intermediate-risk karyotype, whereas Notch1expression lost its predictive value for survival in patients with intermediate-risk karyotype.8. The OS difference between patients with low and high Notch1expression was apparent in the subgroups with high VEGF levels (P=0.001) and high Ang-2levels (P=0.001). However, outcome was much poorer in patients with high than low D114expression in the presence of high VEGF expression (P=0.001). The impact of D114expression on OS was equally prominent in the subgroups of AMI. patients expressing low and high levels of Ang-2(P=0.001).Conclusions1. There was aberrant profile of angiogenesis-related pathway of Notch1/D114, VFGF/VEGFR and Ang/Tie2in newly diagnosed AML patients, such as the up-regulation expression of Notch1, D114, VEGF, VEGFR-2and Ang-2, and abnormal activation of Notch1/D114, and marked increase of VEGFR-2/VEGFR-1and Ang-2/Ang-1expression ratio.2. Correlations of Notch1/D114, VEGF/VEGFR, Ang/Tie2factors was found in untreated AML, and some of the angiogenic-factors was positively associated with age. karyotype and the percentage of leukemic blast in BM. It was suggested that the interaction of Notch1/D114, VEGF/VEGFR and Ang/Tie2signal pathway might play important role in angiogenesis of AML.3. The karyotype and expression of Notch1,D114, VEGF and Ang-2were independent prognostic factors in AML. Additionally, the prognostic value of D114level was more obviously in intermediate-risk cytogenetic AML. Notch1and D114expression had prognostic impact on patients with high VEGF or Ang-2levels. Our data provided evidence that the activation of Notch1pathway may indicate an unfavorable prognosis in AML. Section II The study of Notch pathway in the progression and resistance of chronic myeloid leukemiaBackgroundChronic myeloid leukemia (CML) was a hematological stem cell cloning disorder characterized by Ph chromosome or BCR/ABL fusion gene excessive. It has a progressive course typified by the transition from the chronic phase (CP) to the accelerated phase (AP) and on to blast crisis phase (BP). Most of the patients have reached to complete molecular remission since tyrosine kinase inhibitor(TKI) appeared, but there were still some patients could result in drug resistance and disease progression, and then transit from the chronic phase to the blast phase. The secondary genetic forces responsible for the transition from the chronic phase to the blast phase are not clear.Notch signaling pathway, which consisted of Notch receptors, ligands and cellular effectors, played a key role in regulating cell differentiation, proliferation and apoptosis. Notch signal pathway was first observed in T-cell acute lymphocytic leukemia (T-ALL) with translocation of t(7;9)(q34;q34.3), which brought an activated form of the Notch-1receptor gene, and then promoted the transcription of target genes including the Hesl, HERP and RhoU, the inhibition of T cell differentiation, eventually the occurrence of leukemia. From our previous study, Down-regulation of Notch1could increase T-ALL cells’ chemosensitivity, and it is implied that the abnormal activation of Notch1may take part in drug resistance of T-ALL. Some scholars recently noticed that aberrant activation of Notch1signaling might exist in acute myeloid leukemia (AML), multiple myeloma (MM) and lymphoma. And the effect of Notch1pathway on the progression and drug resistance of CML needed to be further research.ObjectiveTo study the expression of Notch1and Hes1in bone marrow mononuclear cells of CML, to explore the effect of Notch1pathway on the chemosensitivity of K562/A02cells mediated by siRNA, so as to provide the basis for revealing the progression and drug resistance of CML.Methods1. Study samples77adult patients with CML and16healthy adult controls were enrolled in this study.CML patients were divided into four groups:CP group, AP group, BP group and complete remission (CR) group.2.BM mononuclear cells were obtained from CML patients and controls.The total RNA was isolated by Trizol and cDNA was prepared using the M-MLV Reverse Transcriptase. We established real-time quantitative RT-PCR (real-time Q-RT-PCR) methods to detect the mRNA expression of Notch1and Hes1.3. Cell culture K562and K562/A02cell lines were cultured in RPMI medium1640.4. Notch1siRNA transfection K562and K562/A02cells were seeded in24-well plates and after incubation overnight, cells were treated according to the manufacturer’s instructions with Notch1siRNA or control siRNA by Lipofectamine2000.Cells were used for real-time RT-PCR, Western blotting and MTT experiment after72hours siRNA transfection.5. Lysis buffer solubilized the cells and SDS-PAGE fractionated total proteins and transferred onto nitrocellulose membrane. The level of Notch1protein was detected by western blot method in K.562and K562/A02cells.6. Drug sensitivity K562and K562/A02cells were incubated overnight at a density of4×104cells/well in96-well plates, and subsequently transfected with Notch-1siRNA or control siRNA.24h after the transfection, cells were exposed to various doses of doxorubicin for an additional48h and the MTT assay was used to detect live cells. 7. Statistical analyses were performed with the SPSS17.0. Student t test and Mann-Whitney test were used to analyze the difference of the data, and P<0.05were considered significant.Results:1. The Aberrant profile of Notch1mRNA with CML patients The results showed that the Notch1expression were found to be significantly higher in CML BP patients compared to control group (118.4vs.14.1; P=0.002). The Notch1expressions were also significantly higher in CML CP patients compared to control group (68.3vs.14.1; P=0.014). Additionally, no significant difference was found in Notchl expression between BP and CP patients (118.4vs.68.3, P=0.491).2. The expression of Hesl mRNA with CML patients The expressions of Hesl were significantly higher in CML CP patients compared to control group (201.5vs.58.2;P=0.0058). No significant difference was found in Hesl expression between BP patients and control group (285.9vs.58.2;P=0.329) or CP patients (285.9vs201.5,P=0.275).3. The impact on expression of Notchl in K562and K562/A02cells mediated by Notchl siRNA After transfected with Notchl siRNA or control siRNA for72hours, Notch1and Hesl mRNAs were dramatically reduced compared with controls (P<0.05). And Notchl protein levels were also obviously decreased.4. The changes of chemosensitivity of K562and K562/A02cells after down-regulating Notchl expression by siRNA It was found that the IC50value of adriamycin in K562and K562/A02cells were no significant difference between transfected with Notch1siRNA and control siRNA.Conclusions1. There was aberrant activation of Notch1pathway in CML patients, and the expression level of Notch1and Hesl were different from chronic phase to blast crisis, suggesting Notch1pathway might contribute to disease progression.2. Notchl siRNA could effectively down-regulate the expression of Notchl in K562and K562/A02cells. 3. Down-regulation of Notch1mediated by siRNA could not increase sensitivity to adriamycin in K562and K562/A02cells, and it is implied that downregulation of Notch1might have none effect in reversing drug resistance of CML cells. Section III The study of microRNA in drug resistance of acute myeloid leukemiaBackground:Acute myeloid leukemia (AML) was a heterogeneous group of hematopoietic malignancies which severely harmed the health of human. Great progress has been made in the chemotherapy effect with more improved methods, though some patients were failed in chemotherapy because of drug resistance of AML cells either intrinsic or aequired. It was important that we explore the mechanisms of chemo-resistance and efficient methods. MicroRNAs (miRNAs) were short noncoding RNA molecules, which played crucial roles in diverse biological processes,such as cell proliferation, differentiation, apoptosis, tumorigenesis and drug resistance. Recently, it was showed that the expression of miRNAs be aberrant in leukemia. Some miRNAs were recognized as diagnostic and prognostic indicators of leukemia. However, the role of miRNAs in the pathogenesis and drug resistance of AMI. remained unclear, and it needed to further research.ObjectiveTo screen miRNAs which can regulate multidrug resistance of AML,study the expression of miRNAs in AML patients; and investigate the mechanisms of miRNAs in AML pathogenesis and drug resistance, so as to find a new approach for targeted treatment and reversing drug resistance in AMLMethods1. MTT assay was monitored to test the resistance of adriamycin toward K562and drug-resistant K562/A02cells.2. The expression of MDR1was examined by real-time RT-PCR, in order to validate the multidrug resistance of K562/A02cells.3. Microrna microarray was used to detect the miRNA expression profiles in K.562/A02and K562. The four differentially-expressed miRNAs were chosen as candidates.4. Bone marrow samples were obtained from39AML patients. AML patients were divided into newly diagnosed group without any therapy or treatment, completely remission (CR) group and relapsed/refractory group according to the response to chemotherapy. BM mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation.5. TRIzol reagent was applied to extracte total RNA from cells and RT kit performed for reverse transcription. Real-time RT-PCR was used to examine the4miRNAs in AML patients.6. The let-7a fragment and negative control were subcloned into pRNAT-U6.1/Hygro. The pRNAT-let-7a was confirmed by restriction analysis and DNA sequencing.7. Lipofectamine2000was used to transfected the let-7a expression vector and negative control into AML cell lines. Stable transfectants were selected by Hygromycin B.8. Lipofectamine2000was applied to transfected miR-23a mimic and negative control into K562/A02cell lines. Real-time RT-PCR was used to detect the miR-23a lever after48h transfection.9. Serial dilutions of doxorubicin were added into cells in96-well culture plates for48h. MTT method was used to detect the drug sensitivity, and IC50values were calculated after correction for background absorbance.10. Apoptosis assays by flow cytometry:Cells were treated with doxorubicin. The apoptotic cells were analyzed by flow cytometry using Annexin V and propidium iodide (PI) after48hours.11. The expression of MDR1and MCL1were detected by real-time RT-PCR in K.562/A02cell lines after transfected miR-23a mimic and negative control, and β-actin was detected as internal control.12. SPSS software17.0wad applied to statistical analysis. T test and Mann-Whitney test was used to compare median miRNA expression in three groups of AML patients. P<0.05was recognized significant.Results1. MTT assays showed that the IC50value of K562/A02cells was18.79±1.72μg/ml, and the IC50value of K562cells was (0.22±0.03μg/ml), which was statistically significant (P<0.05), indicating that K.562/A02cells were resistant to adriamycin.2. P-actin was used as internal control for MDR1mRNA expression. The MDR1expression in K562/A02was significantly higher than that of K562cells (1.24±0.07vs8.19±2.74×10-6)(P<0.05).3. K562and K562/A02cells were profiled for miRNA expression using the Exiqon miRNAarray platform. Of the384genes covered by Exiqon miRNAarray,9miRNAs were found down-regulated more than twofold in K562/A02when compared with K562. While2miRNAs were up-regulated more than twofold in K562/A02cells than in K562. We selected4significantly differential miRNAs (let-7a, miR-23a, miR-663and miR-144) as candidate miRNAs.4. MiRNAs expression in patients:MiR-144was down-regulated in the AML samples of newly diagnosed compared to completely remission AML patients (P<0.05). Although no significant difference was found in miR-23a levels between the newly diagnosed AML patients and completely remission patients, there was an obvious trend that the newly diagnosed AML patients expressed lower levels of miR-23a compared with completely remission AML patients (P<0.05). There was no difference in let-7a and miR-663levels in the three groups of patients.5. The restriction analysis and DNA sequencing showed that the let-7a expression vector was correct.6. We observed that about95%percent of K562/A02cells transfected with let-7a expression vector stable express GFP after4weeks.7. MTT results found that the IC50value of adriamycin in K562/A02cells transfected with let-7a stable expression vector were14.64±2.34μg/ml, while transfected with control were14.19±1.36μg/ml, and there was no significant difference (P=0.846).8. Real time RT-PCR showed that miR-23a levels were significantly up regulated in K562/A02cells transfected with miR-23a mimic (P<0.01).9. From the result of flow cytometry, we found that the apoptosis rate of K562/A02 cells transfected with miR-23a mimic obviously higher than control cells (30.65±1.11%vs.14.46+1.5%, P<0.05).10. It was showed that the expression of MDR1in K562/A02cells transfected with miR-23a mimic was significantly decreased than those of the control (P<0.05), however, there was no significant different in the lever of MCL1(P=0.18).Conclusions1. There were different expression of miRNAs in K562/A02cells compared with K562cells, indicated that the miRNAs might take part in the drug resistance in AML.2. MiR-144was down-regulated in newly diagnosed AML and miR-23a was down-regulated in relapse/refractory AML patients. The results indicated that miR-144and miR-23a may play important roles in the pathogenesis and drug resistance of AML.3. Let-7a expressing vector and K562/A02cells stably expressing Let-7a were successfully established, however, up-regulation of Let-7a could not increased chemo-sensitivity of K562/A02cells.4. The overexpression of miR-23a dramatically promoted drug-induced apoptosis in K562/A02cells, and the decrease of MDR1.MiR-23a might be one of the new methods to reverse drug resistance of AML...