Functional Studies Of MiRNAs Associated With Drug Resistance In Human Acute Myeloid Leukemia And Their Mechanisms

Background:Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis characterized by the uncontrolled proliferation and accumulation of immature and dysfunctional hematopoietic progenitors accompanied by blockage in normal hematopoiesis. Despite the tremendous progress in AML treatment achieved during the last decades, the5-year survival rate of patients with AML remains relatively low. Drug resistance still accounts for most of the tumor relapses in chemotherapy-treated AML patients. In general, there are two classes of drug resistance: inherent (natural) resistance and acquired resistance. Many factors account for the occurrence of chemotherapeutic multidrug resistance (MDR), including overexpression of drug resistance-related proteins, alterations in drug targets, escape from cell cycle checkpoints, altered pharmacokinetics, increased drug efflux, and stem cell development. Given the pressing need to improve outcomes in AML patients, it is crucial to elucidate the mechanisms of chemoresistance.MicroRNAs (miRNAs, miRs) represent a new class of small, noncoding endogenous RNAs that range in size from19to25nucleotides (nt) and can negatively regulate target gene expression at the post-transcriptional level. Mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) to cause either degradationor inhibition of translation by binding to the3’-untranslated region (3’-UTR) of target mRNAs. Due to their diverse functions in cell proliferation, apoptosis, invasion, cell differentiation, cell cycle progression, and hematopoiesis, overwhelming evidence has indicated the important regulatory roles of miRNAs during carcinogenesis and chemoresistance. Current data suggest that microRNAs can be categorized as oncogenic or tumor-suppressive based on their functions and targets. However, the mechanism of microRNA-mediated drug resistance in AML is not fully understood. Therefore, the identification of miRNAs function highlights a new approach for the development of drug resistance therapy in AML, which is of great importance for the enhancement of AML treatment efficacy. Objective:The aim of this study was to identify the miRNAs which involved in the AML chemoresistance; Furthermore, we assessed the expression of miRNAs in patients with AML and analyze their expression relationship and clinical significance; Additionally, we intended to investigate the biological effects and determine the roles of miRNAs in multidrug resistance of AML; We will further elucidate the underlying molecular genetic basis of miRNAs in AML chemoresistance. Our original research will provide new mechanisms and putative biomarkers of sensitivity to chemotherapeutics in the relapsed/refractory AML patients, which is of great importance for the enhancement of AML treatment efficacy.Materials and methods:1. Microarray experiments: Applied Biosystems TaqMan Low Density Array was used for detect miRNA expression profile between AML drug-resistant cell lines and sensitive AML cell lines.2. Patient samples: Bone marrow samples from120AML patients and40normal donors were obtained after informed consent at the Qilu Hospital, Shandong University. Study protocols were approved by the Medical Ethical Committee of Qilu Hospital, Shandong University. Mononuclear cells (MNCs) were isolated from bone marrow aspirates by density-gradient centrifugation using Ficoll-Paque Plus and stored at-80℃.56AML patients were newly digagnosed and44AML patients were in complete remission (CR),20AML patients were in relapsed/refractory.3. Real time RT-PCR: Total RNA was extracted from AML cells and patient samples using mirVanaTM miRNA Isolation kit. Reverse transcription was performed with TaqMan MicroRNA Reverse Transcription (RT) kit. In order to validate their expression changes detected in microarray assay, the expression of mature miR-29b was measured using a TaqMan MicroRNA Assay.4. The biological effects of miR-29b, AF1q and CD44in MDR of AML. 4.1Transfection: AML cell lines were transfected with synthetic miR-29b mimic or miR-29b inhibitor using Lipofectamine2000reagent according to the manufacturer’s instructions. Real time RT-PCR was applied to detect the expression of miR-29b.4.2AF1q lenti virus production and transduction: Packaging of the AF1q constructs in pseudoviral particles according to the manufacturer’s instructions. AML cells were stably transfected with pLOC-EGFP-AF1q shRNA or pLOC-EGFP-AF1q, and maintained under blasticidin selection. Empty lentivirus was used as a control for the experiments.4.3Construction of CD44shRNA plasmid: The CD44-shRNA plasmid was transfected into AML cells. The expression of CD44was detected by Real time RT-PCR and Western blot after transfection with the CD44-shRNA plasmid for48h.4.4Cell viability assay: Cells were treated with serial dilutions of doxorubicin (DOX), cytarabine (Ara-C) or darubicin (IDA) for48h. The inhibition of cell growth was analyzed by CCK8assay, and IC50values (the concentration of drug that is required for50%inhibition of cells) were calculated after correction for background absorbance.4.5Apoptosis assay: After treatment with DOX, Ara-C or IDA for48h, The AML cells were stained with Annexin V/FITC followed by PI. Then cells were immediately measured by FACS Calibur.5. The mechanisms of miR-29b/AF1q/CD44axle in AML chemoresistance.5.1We performed abioinformatics search (Targetscan, Pictar) for the prediction of the miR-29b target genes. Real time RT-PCR and Western blot were applied to detect the effect of altered miR-29b expression on AF1q.5.2Dual luciferase activity assay: We construct luciferase reporter vectors carrying wild-type or mutated AF1q3’-UTR target sites and cotransfected these vectors with the miR-29b mimic into293T cells. Firefly and Renilla luciferase activities were determined using the dual luciferase reporter assay system.5.3The expression of AF1q and CD44were evaluated in AML cells and BM mononuclear cells from AML patients by Real-time RT-PCR and Western blot, The statistic analysis was used to evaluate the correlation between the above genes studied, and the association with clinical features. 5.4Collect the stable transfected pLOC-EGFP-AF1q shRNA or pLOC-EGFP-AFlq AML cells and detect the CD44expression by Real time RT-PCR and Western blot.5.5The miR-29b mimic or miR-29b inhibitor were transfected into AML cells. The expression of CD44was detected by Real time RT-PCR and Western blot after transfection with miR-29b mimic or miR-29b inhibitor for48h.5.6After treatment with DOX for12h,24h,48h, the AF1q and CD44expression were detected by Real time RT-PCR and Western blot assays.5.7The groups were as follows:①miR-29b mimic;②miR-NC;③pLOC-EGFP-AF1q;④pLOC-EGFP-NC;⑤miR-29b mimic+pLOC-EGFP-AF1q;⑤miR-29b mimic+pLOC-EGFP-NC;⑥miR-NC+pLOC-EGFP-NC. The inhibition of cell growth was analyzed by CCK8assay and CD44expression was evaluated by Western blot.5.8Dual luciferase activity assay was employed to detect the the effect of altered AF1q expression on CD44promoter activity.5.9Co-IP assay was applied to detect the combination between AF1q and TCF7L2.5.10CHIP assay was used to detect the binding sites of TCF7L2in the promoter of CD44.6. Statistical analysis: Statistical analysis was carried out using SPSS software (version17.0). Data are expressed as means±standard errors of at least3independent experiments. Student’s t tests and one-way analysis of variance were used to determine significance between groups. Differences with P-values of less than0.05were considered statistically significant.Results:1. MiRNA microarray results:A total of14miRNAs were upregulated and10were significantly down-regulated in AML drug-resistant cell lines, on the basis of a threshold of a median fold change greater than2.0. We selected the most significantly different miRNAs (miR-29b and miR-181b) to further study their roles in AML chemoresistance.2. A significant downregulation of miR-29b were observed in AML drug-resistant cells compared with that in parental AML susceptible cells.3. Aberrant expression profile of miR-29b, AF1q and CD44in AML patient samples. 3.1The expression of miR-29b in newly diagnosed group and relapsed/refractory group was significantly lower than that in controls; miR-29b was significantly elevated in CR group compared with newly diagnosed group and relapsed/refractory group; miR-29b was downregulated in AML samples from relapsed/refractory patients in comparison with those of newly diagnosed AML patients; There were no differences in miR-29b levels between the CR group and controls.3.2AF1q was significantly elevated in newly diagnosed group and relapsed/refractory group compared with controls; The expression of AF1q in newly diagnosed group and relapsed/refractory group were significantly higher than that in CR group; AF1q was upregulated in AML samples from relapsed/refractory patients in comparison with those of newly diagnosed AML patients; There were no differences in AFlq levels between the CR group and controls.3.3CD44was upregulated in AML samples from newly diagnosed group and relapsed/refractory group in comparison with those of controls; The expression of CD44in newly diagnosed group and relapsed/refractory group was significantly higher than that in CR group; CD44was significantly elevated in relapsed/refractory group compared with newly diagnosed group; There were no differences in CD44levels between the CR group and controls.4. The roles of miR-29b, AF1q and CD44in AML chemoresistance.4.1Real time RT-PCR confirmed that miR-29b mimic effectively enhanced the expression of mR-29b; The miR-29b inhibitor can decrease the expression of miR-29b effectively.4.2Realtime RT-PCR and Western blot confirmed that pLOC-EGFP-AF1q adenovirus infection effectively enhanced the expression of AF1q; The pLOC-EGFP-AFlq shRNA adeno virus infection can decrease the expression of AF1q effectively.4.3Real time RT-PCR and Western blot confirmed that CD44shRNA plasmid effectively inhibited the expression of CD44.4.4Overexpression of miR-29b5knockdown of AF1q or CD44increased the sensitivity of AML chemoresistant cells to cytotoxic chemotherapeutic agents; Inhibition of miR-29b or overexpression AF1q decreased the sensitivity of AML cells to cytotoxic chemotherapeutic agents. 4.5Ectopic expression of miR-29b, inhibition of AF1q or CD44markedly increased chemotherapy-inducing apoptosis (as measured by the percentage of Annexin V-FITC-positive cells) in AML drug-resistant cells; Knockdown of miR-29b or ectopic expression of AF1q markedly decreased chemotherapy-inducing apoptosis.5. The mechanisms of miR-29b/AF1q/CD44axle in AML chemoresistance.5.1The database Target Scan Human6.2predict AF1q as potential target of miR-29b; the ectopic expression of miR-29b in K562/A02and HL60/ADM cells robustly suppressed endogenous AF1q expression; Conversely, knockdown of miR-29b markedly increased the expression of AF1q.5.2Transfection with the miR-29b mimic significantly decreases luciferase activity, whereas mutation of the3’-UTR binding sites of AF1q in the reporter vector abrogated this effect, indicating that miR-29b directly interacted with the3’-UTR of AF1q.5.3Real-time RT-PCR and Western blot showed that the expression of AF1q and CD44was much higher in AML drug-resistant cells compared with that in parental AML susceptible cells. Spearman correlation analysis showed that AF1q and CD44both displayed negative associations with miR-29b in AML patients, AF1q exhibited a positive correlation with CD44in AML patients.5.4The expression level of CD44decreased concurrently with the AF1q after we transfected K562/A02cell with AF1q shRNA and visa verse.5.5Ectopic expression of miR-29b led to CD44downregulation, while inhibition of miR-29b led to upregulation of CD44.5.6Enforced expression of oncogene AF1q partially rescues the miR-29b mediated CD44downregulation; CCK8assay showed that IC50value was partially rescued by up-regulation of the AF1q.5.7After treatment with DOX for12h,24h,48h, the AF1q and CD44expression were decreased significantly.5.8Luciferase assay show that AFlq directly transcriptional activates CD44by binding to the CD44promoter.5.9Co-IP assay validated that AF1q can interact with TCF7L2.5.10CHIP assay validated that TCF7L2bind to the CD44promoter. Conclusion:1. miRNAs were differentially expressed in AML sensitive and drug-resistant cells, indicating that miRNA may play an important role in the development and maintenance of MDR in AML.2. Restoring miR-29b expression greatly enhanced the sensitivity of AML drug-resistant cell lines to cytotoxic chemotherapeutic agents and increased chemotherapy-inducing apoptosis.3. The effect of miR-29b/AF1q/CD44regulatory axle in MDR of AML suggests a therapeutic potential for relapsed/refractory AML patients.4. AF1q directly transcriptional activates CD44via interaction with TCF7L2. Objective:The aim of this study was to detect the expression of miR-181b in AML cells to verify the results of microarray; To measure miR-181b expression level in patients with AML and analyze their expression relationship and clinical significance; To investigate the biological effects of miR-181b and determine the roles of miR-181b in multidrug resistance of AML; To clarify the underlying regulatory mechanisms of miR-181b in multidrug resistance of AML.Methods:1. Bone marrow samples from AML patients and normal donors were obtained after informed consent at the Qilu Hospital, Shandong University. Mononuclear cells (MNCs) were isolated from bone marrow aspirates by density-gradient centrifugation using Ficoll-Paque Plusand stored at-80℃. AML patients were classified into newly-diagnosed group(31), CR group(37) and relapsed/refractory group (12) according to the response to chemotherapy.2. Total RNA was extracted from AML cellsusing mirVanaTM miRNA isolation kit. Reverse transcription was performed with TaqMan MicroRNA Reverse Transcription (RT) kit. The TaqMan MicroRNA Assay was used to validate miR-181b expression changedetected in microarray assay. All qRT-PCR was conducted on the AppliedBiosystem7900HT System and performed in triplicates.3. The expression of mature miR-181b in AML patients were measured using the TaqMan MicroRNA Assay and were assessed the clinical significance.4. The roles of miR-181b in AML multidrug resistance.4.1AML cell lines were transfected with synthetic miR-181b mimic or inhibitor by using Lipofectamine2000reagent. Real time RT-PCR was applied to detect the expression of miR-181b after transfected for48h.4.2Proliferation assay:Cells were treated with serial dilutions of DOX, Ara-C or IDA for48h. The inhibition of cell growth was analyzed by CCK8assay, and IC50values were calculated. 4.3Apoptosis assay: After treatment with DOX, Ara-C or IDA for48h, The AML cells were stained with Annexin V/FITC followed by PI. Then cells were immediately measured by FACS Calibur.5. Prediction and validation of the target genes of miR-181b.5.1Target scan and Pictar were used for the prediction of the miR-181b target genes.5.2Real time RT-PCR and Western blot were applied to detect the expression of HMGB1and Mcl-1after miR-181b was up-regulated or down-regulated.5.3We construct luciferase reporter vectors carrying wild-type or mutated HMGB1or Mcl-13’-UTR target sites and cotransfected these vectors with the miR-181b mimic into293T cells. Dual luciferase activities were determined to validate the effect of miR-181b on target genes.6. The roles of HMGB1in AML multidrug resistance.6.1Real-time RT-PCR and Western blot were applied to detect HMGB1epression in BM mononuclear cells from AML patients. The statistic analysis was used to evaluate the correlation with miR-181b expression and clinical features.6.2Real-time RT-PCR and Western blot were applied to detect HMGB1epression after transfected with HMGB1siRNA for48hours.6.3The inhibition of cell growth was analyzed by CCK8assay after HMGB1siRNA was transfected for48hours.6.4After treatment with DOX, Ara-C or IDA for48h, The AML cells were stained with Annexin V/FITC followed by PI. Then cells were immediately measured by FACS Calibur.7. Statistical analysis: Student’s t test was used for statistical comparison between groups. P values less than0.05were considered statistically significant.Results:1. A significant downregulation of miR-181b was observed in AML MDR cells compared with that in parental AML susceptible cells.2. The expression of miR-181b in newly diagnosed group and relapsed/refractory group was significantly lower than that in controls; miR-181b was significantly elevated in CR group compared with newly diagnosed group and relapsed/refractory group; miR-181b was downregulated in AML samples from relapsed/refractory patients in comparison with those of newly diagnosed AML patients; There were no differences in miR-181b levels between the CR group and controls. 3. The roles of miR-181b in AML multidrug resistance.3.1Real time RT-PCR results showed that miR-181b mimic effectively enhanced the expression of miR-181b; The miR-181b inhibitor can decrease the expression of miR-181b effectively.3.2Overexpression of miR-181b increased the sensitivity of leukemia cells to cytotoxic chemotherapeutic agents.3.3Ectopic expression of miR-181b markedly promoted chemotherapy-inducing apoptosis in AML drug-resistant cells.4. Prediction and validation of the target genes of miR-181b.4.1HMGB1and Mcl-1were predictedas potential targets of miR-181b by using Target Scan Human6.2.4.2The ectopic expression of miR-181b in AML chemoresistan cells robustly suppressed endogenous HMGB1and Mcl-1expression; Conversely, knockdown of miR-181b markedly increased HMGB1and Mcl-1expression.4.3Transfection with the miR-181b mimic significantly decreases luciferase activity, whereas mutation of the predicted binding sites of HMGB1or Mcl-1in the reporter vector abrogated this effect, indicating that miR-181b directly interacted with the3’-UTR of HMGB1or Mcl-1.5. The roles of HMGB1in AML chemoresistance.5.1HMGB1was significantly elevated in newly diagnosed group and relapsed/refractory group compared with controls; The expression of HMGB1in newly diagnosed group and relapsed/refractory group were significantly higher than that in CR group; HMGB1was upregulated in AML samples from relapsed/refractory patients in comparison with those of newly diagnosed AML patients; There were no differences in HMGB1levels between the CR group and controls.5.2A significant up-regulation of HMGB1were observed in AML chemoresistant cells compared with that in parental AML sensitive cells.5.3Real time RT-PCR and Western blot confirmed that HMGB1siRNA effectively inhibited the expression of HMGB1.5.4Knockdown of HMGB1increased the sensitivity of AML chemoresistant cells to cytotoxic chemotherapeutic agents. 5.5Inhibition of HMGB1markedly increased chemotherapy-inducing apoptosis (as measured by the percentage of Annexin V-FITC-positive cells) in AML drug-resistant cellsConclusions:1. miR-181b was decreased significantly in human multidrug resistant AML cells and relapsed/refractory AML patient samples, indicating that miR-181b functioned as a tumor suppressor in AML chemoresistance.2. Overexpression of miR-181b increased the sensitivity of AML cells to cytotoxic chemotherapeutic agents and promoted drug-induced apoptosis.3. Enforced expression of miR-181b could enhance drug sensitivity and apoptosis in AML multidrug resistant cells at least partially though direct suppression of its target genes, HMGB1and Mcl-1.