| Part ? Bioinformatics analysis and experimental verification of MV in the CML associated malignant transformationObjective:Microvesicles (MV) are functional bubbles derived from plenty of cells. MV plays critical roles in the progression and relapse of cancers. In our previous study, we developed a model to transform normal hematopoietic transplants to leukemia-like cells by K562-MVs. Here, we elucidated the transformation mechanism of this model through analyzing the gene expression and constructing co-regulatory networks of transcription factors (TFs) and miRNAs.Methods:Supernatant of human CML blast crisis cell line K562 was collected to isolate MV by gradient centrifugation. To determine the gene expression change and the mechanism of the process that K562-MVs transform MNCs, we performed RNA-seq and small RNA-seq for five samples of the key time points, which were samples of K562-MVs, MNCs,1 week(1W)/2 weeks (2W)/3 weeks (3W) cells after MVs incubation. We used the in-house scripts with Poisson distribution to test the differentially expressed genes and miRNAs between different samples. We obtained 161 differentially expressed TFs in the transformation process. Finally,42 key TFs were selected based on their importance to the hematopoietic system and cancer regulation. The differentially expressed miRNAs and genes were hierarchically clustered using an average linkage algorithm and a Euclidean distance for the distance measure. We utilized MeV to visualize the clustered data. To confirm the effect of miR-146b, K562 cells were transfected with miR-146b mimics and inhibitor. DNA double-strand breaks (DSBs) were assessed by agarose gel electrophoresis using genomic DNA isolated from recipient cells. Intracellular reactive oxygen species (ROS) were detected using a Reactive Oxygen Species Assay Kit.Results:We have identified 3,717 differentially expressed genes (DEGs) (including 161 TFs) and 143 differentially expressed miRNAs (DEMs) in the transformation process. To explore the perturbed mechanisms of cancer pathways, we constructed the regulatory network among miRNAs, TFs, and genes in cancer pathways. We found that, at the beginning, anti-tumor miRNAs were increased and several defense pathways were initiated. Later, oncomiRs and genes involved in the cell cycle, DNA repair, and energy metabolism pathways were up-regulated. Expression and regulatory network analyses revealed that a number of TFs and miRNAs were responsible for the pathway dysregulation and transformation, especially regulators highly expressed in MVs (e.g., miR-146b). Our experiments revealed that a significantly increased level of miR-146b was observed in K562-MVs after transfecting miR-146b mimics into the K562 cells, while a decrease could be found when transfected with miR-146b inhibitor. Up-regulation of miR-146b in K562 could accelerate the transformation process via targeting NUMB, accompanied by the increasing of ROS level and genome instability of the recipient cells. Elevated levels of NUMB in recipient cells, a significant decay of transformation was observed.Conclusion:In summary, using expression and regulatory network analysis, we explored the potential transformation mechanism for our previous model that K562-MVs transforming MNCs to leukemia-like cells. We identified miR-146b, as a downstream regulator of BCR-ABL1, promoted the transformation by targeting several important genes to affect genome instability and cell proliferation. Our study provided a meaningful insight into the CML BC transformation and donor cell leukemia, as well as an opportunity for studying the changes of normal cells to cancer-like cells.Part ? Role of microvesicles in cessation of TKI in CML patientsObjective:Increasing numbers of CML patients who remain on tyrosine kinase inhibitors (TKIs) for years have undetectable minimal residual disease (UMRD). Whether TKIs could be safely discontinued is one of the key points of chronic myelogenous leukemia (CML) currently. The main obstacles were that there appear to be no exact patients or disease characteristics that identify in advance those who can safely discontinue TKIs at present, nor excellent biomarker was found in the monitoring.Methods:Before this work, we have viewed 1057 patients with a confirmed diagnosis of CML in chronic or accelerated phase and treated with ongoing TKIs at the Union Hospital and Tongji Hospital. Only 22/1057 patients (2.2%) had discontinued TKIs therapy and were analyzed in this work.10ml of bone marrow samples were obtained from each patients following informed consent when they were about to cessation. Mononuclear cells (MNCs) were extracted from the bone marrow. The phenotype of CML-LSC was analyzed by flow cytometry using fluorescein is thiocyanate-labeled antibodies to CD45, CD34, CD38 and CD26. MDSC was also detected by FACS. NOD/SCID mice of 3-4-week-old were used as recipients of bone marrow from the patients. Mononuclear cells were extracted from 10 ml bone marrow of each patient and injected into two individual mice from the tail vein.Results:The median age of these 22 patients was 25 ys (range 14-68 ys). The male:female ratio was 8:14.20/22 patients were in chronic phase and 2/22 was in the accelerated phase at diagnosis. Molecular recurrence was detected in 10/22 patients (50%) until now. No disease progression and none morbidity were observed after cessation. TKI treatment was restarted immediately in 6 patients who all then recovered to MMR with an average of 5.33 months (range 3-9 months). The remaining 4 patients refused to restart TKIs therapy and have lost the sustained MMR for 2 months until now. No significant difference was found in median course of imatinib therapy between the TRF group and the group of molecular relapse (70.5±7.7 vs 76.7±6.3, P= 0.54). Similarly, time to MMR (10.3±1.6 vs 7.5±1.4, P= 0.21), age (29.2±4.3 vs 36.4±6.2, P=0.34), gender and Sokal risk also showed no difference between the two groups. CD34+CD38-CD26+ cells could be detected in 20/22 patients even they have achieved UMRD for years, indicating that CML-LSC could not be eliminated by TKIs. However, no significant difference was observed in the number of CD34+CD38-CD26+ cells (0.27%±0.07 vs 0.24%±0.07, P> 0.05) between the TRF group and the relapse group. About 30 days after tail vein injection,9/44 mice presented obvious less activity and low weight; and there were similar presentation in mice transplanted with K562 cells as a control (n= 5). Spleen enlargement were observed when autopsy in 6/9 mice transplanted with MNCs from the bone marrow of patients. Leukemia-like malignant cells could be observed in bone marrow, spleen, liver and kidney of the mice, which was confirmed by immunohistochemistry assay of human anti-CD45 and anti-CD38 antibodies. The bone marrow responsible for the mice leukemogenesis came from 5 patients (No.3,4,11,12,17 for 7 mice) suffered molecular recurrence and 2 (No.21) in the TRF group. Human BCR-ABL1 mRNA could also be detected in the bone marrow and the spleen. In the monitoring, we discovered that BCR-ABLl copy numbers in MV significantly varied between CML patients in HR, CCyR, MMR and CMR. For the 22 patients who stopped TKIs, BCR-ABLl in MV was significantly lower in the group of TFR (1.15 ± 0.15 vs 1.91 ± 0.35, P= 0.47).Conclusion:In summary, this is the first report of the cessation data about Chinese CML patients. We investigated the connection of CML-LSC and the post-cessation relapse. Whether the patient would relapse seems to depend on the function but not the number of LSC. Xenograft model in mice would provide a novel and useful model to analyze the function of LSC and predict the relapse. These data of MV detection probably improved the sensitivity of leukemic cell detection and provided important information about optimal molecular monitoring schedules in TKI discontinuation strategies. |
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