The Roles Of MCL1, AF1q And MicroRNA In Human Leukemia And Their Mechanisms

Section IThe study of MCL1 gene in drug resistance of human leukemiaBackground:Leukemia is a class of hematological malignancies which seriously impacts human's health, characterized by abnormal proliferation of clonal leukemic hematopoietic precursor cells and impaired production of normal hematopoiesis. Although different strategies have been adopted to obtain complete remission (CR), about 60～70% of patients fail in chemotherapy or relapse. The major reason is multi-drug resistance (MDR). Various mechanisms are involved in MDR pathogenesis. Abnormalities in the apoptotic response play an important role in the development of drug resistance by leukemic cells.The myeloid cell leukemia sequence 1 (BCL2-related) gene (MCL1) is an anti-apoptotic member of the BCL2 family. Similar to BCL2 and BCL2L1, MCL1 can interact with BAX and BAK1 to inhibit mitochondria-mediated apoptosis. MCL1 has been shown to be up-regulated in numerous hematological (e.g. chronic lymphocytic leukemia (CLL)) and solid tumor malignancies (e.g. hepatocellular carcinoma and non-small cell lung cancer). High level expression of MCL1 also correlates with high tumor grade and poor survival in patients. Recently a study showed that expression of MCL1 was significantly associated with prednisolone sensitivity in childhood acute lymphoblastic leukemia (ALL), which indicates that MCL1 may play an important role in MDR of leukemia.Objective:To investigate the expression of MCL1 in patients with acute leukemia; To study the effect of MCL1 overexpression on drug sensitivity and drug induced apoptosis in leukemic cells; To reveal the pathogenesis of MCL1 in MDR and provide the basis for molecular targeted therapy.Materials and methods:1. Patient samples:Bone marrow samples were obtained from 47 patients with AML and ALL.27 patients were newly digagnosed/relapsed/refractory and 20 patients were with CR. Ficoll-Hypaque density gradient centrifugation was used to separate mononuclear cells. Real time RT-PCR was used to detect the expression of MCL1.2. Construction of MCL1 expression plasmid:The MCL1 mRNA CDS fragment was amplified from multi-drug resistant cell line K562/A02 by RT-PCR. The PCR fragment was inserted into the pLXSN vector and the resultant plasmid was designated pLXSN-MCL1.3. Stable transfection:The pLXSN-MCL1 plasmid was transfected into K562, HL60 and Jurkat cells.48 hours after transfection, cells were selected in G418 containing medium for at least 2 weeks. The expression of MCL1 was detected by Real time RT-PCR and Western blot.4. Quantitative real time PCR:Total RNA was extracted from cells using TRIzol reagent and reverse transcription was performed with M-MuLV reverse transcriptase. Quantitative real time PCR was used to detect MCL1 mRNA levels.5. Western blot:Cells were solubilized in RIPA lysis buffer and cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis. Western blot method was used to detect MCL1 protein levels.6. Drug sensitivity:Cells were seeded onto 96-well culture plates and treated with serial dilutions of doxorubicin for 72 hours.3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method was used to detect live cells. IC50 values, defined as the concentration inducing 50% loss of cell viability, were calculated.7. Apoptosis assays by flow cytometry:Cells were cultured on 6-well plates and treated with doxorubicin. After drug treatment for 72 hours, the apoptotic cells were analyzed by flow cytometry using Annexin V and propidium iodide (PI).Results:1. MCL1 expression in patients:In the 27 newly diagnosed/relapsed/refractory leukemia patients,13 patients (48%) expressed high levels of MCL1. In the 20 patients with CR, only 4 patients (20%) expressed high levels of MCL1.2. The pLXSN-MCL1 plasmid and its control were transfected into leukemia cell lines K562, HL60 and Jurkat. Real time RT-PCR showed the MCL1 mRNA levels were increased by 3.3,6.3,9.6-fold respectively after transfection. Western blot showed MCL1 protein levels were also significantly up-regulated, which was consistent with the results of quantitative real time RT-PCR.3. Overexpression of MCL1 decreased sensitivity of leukemia cells to doxorubicin: MTT results showed that after transfection of pLXSN-MCL1, IC50 values for K562, HL60 and Jurkat cells were significantly higher than those for control cells (K562:103.05±5.79 versus 40.6±2.9μg/L, HL60:58.11±0.92 versus 18.14±1.07μg/L, Jurkat:53.12±0.64 versus 23.27±0.75μg/L, respectively).4. MCL1 overexpression inhibited drug-induced apoptosis in leukemia cells:Flow cytometry showed that K562, HL60 and Jurkat cells transfected with MCL1 expression plasmid underwent less apoptosis than control cells (K562:44.18±4.54% versus 87.69±5.77%,HL60:20.81±6.17% versus 45.46±2.56%,Jurkat: 29.56±2.82% versus 70.91±9.30%, respectively).Conclusion:1. MCL1 expression is higher in patients with newly diagnosed, relapsed and refractory leukemia than in patients with complete remission. 2. MCL1 overexpression decreases sensitivity of leukemia cells to cytotoxic drugs.3. MCL1 overexpression inhibits drug-induced apoptosis in leukemia cells.4. MCL1 plays an important role in drug resistance of leukemia and may be a new target in leukemia therapy.SectionⅡMicroRNA-29b regulates AFlq expression in human leukemiaBackground:Leukemia is the most common hematological malignancy which seriously impacts human's health. Various machanisms are involved in leukemia pathogenesis. Many patients with leukemia carry chromosomal abnormalities which is an important reason for the disease. The AF1q gene, also named MLLT11, was initially identified as a mixed-lineage leukemia (MLL) fusion partner from two acute myeloid leukemia (AML) patients with t(1;11)(q21;q23) chromosomal abnormality. The expression of AF1q is found to be elevated in AML and ALL patients. High AF1q expression is a poor prognostic marker for pediatric AML, adult normal cytogenetics AML and myelodysplastic syndrome (MDS). However, the mechanism of AF1q expression regulation remains unclear.MicroRNAs (miRNAs) are recently found as a class of small, non-protein-coding RNA molecules that are only 19-25nt long and are well conserved in eukaryotic cells. Mature miRNA can bind to the three prime untranslated regions (3'UTR) of target mRNAs usually resulting in gene silencing and translational repression. Now there are almost 700 miRNAs in human and each miRNA can regulate about 200 genes. So it is estimated that over 30% of all human genes are targeted by miRNAs. MiRNAs are found to be up or down regulated in leukemia patients. Some miRNAs are involved in the growth, differentiation, proliferation or apoptosis of leukemic cells. Some miRNAs are recognized as prognostic biomarkers for human leukemia. Thus, we hypothesized that AF1q expression might also be regulated by miRNAs.Objective:To find out the miRNA which can regulate the expression of AF1q in leukemia; To study the role of miR-29b in regulation of AF1q expression and its mechanism; To find a new prognostic biomarker for AML.Materials and methods:1. Computational prediction of miRNA:TargetScanHuman and miRGen were used to identify miRNA candidates that may potentially regulate AF1q.2. Patient samples:Bone marrow samples were obtained from 56 AML patients. Ficoll-Hypaque density gradient centrifugation was used to separate mononuclear cells. Used microarray and real time PCR to detect the expression of miR-29b and AF1q in patient samples.3. Construction of GFP reporter vectors and transfection:The AF1q 3'UTR fragment and its mutation of the miR-29b binding site in AF1q 3'UTR were subcloned into pEGFP-C1. New constructs were named pGFP-A3U and pGFP-A3U-M and were transfected into H157 cells.4. Mimic transfection:miR-29b mimic and negative control mimic were transfected into a leukemia cell line (REH) and two lung cancer cell lines (H157 and SKMES1) using LipofectamineTM 2000.72h after transfection, cells were used for real time RT-PCR and Western blot.5. Real time RT-PCR:Total RNA was extracted from cells using TRIzol reagent and reverse transcription was performed with RT kit. Real time PCR was used to detect the expression of AF1q, GFP andβ2M.6. Western blot:Cells were solubilized in GLB lysis buffer. Total proteins were separated by 4-12% NuPAGE gel electrophoresis. Western blot method was used to detect AF1q protein level. Results:1. Through using computer system, we identified that miR-29a/b/c potentially regulate AF1q expression.2. Microarray result showed that most of the 56 AML patients expressed high levels of AF1q and low levels of miR-29b which meant AF1q and miR-29b expression were inversely related in AML. Patients with low miR-29b have poorer overall survival.3. MiR-29b mimic and negative control mimic were transfected into REH, H157 and SKMES1 cells. Real time RT-PCR showed that AFlq mRNA levels were significantly down regulated in cells transfected with miR-29b mimic. Western blot showed that AF1q protein levels in miR-29b transfected cells were also lower than those of control cells.4. To test whether miR-29b could specifically regulate AF1q expression, we transfected pGFP-A3U and pGFP-A3U-M into H157 cells. Results showed that GFP expression was significantly lower in cells transfected with AF1q 3'UTR than in cells transfected with the mutation of miR-29b binding site in AF1q 3'UTR.Conclusion:1.MiR-29b and AF1q expression are inversely related in AML patients.2. AF1q expression is regulated by miR-29b in leukemic cells.3. MiR-29b regulates AF1q expression by interacting with the 3'UTR of AF1q gene.