Increased Expression of HERG1 K+ Channels Contribute to MDS Progression and Display Correlation with Prognosis Stratification

Abstract Background: Myelodysplastic syndromes (MDS) are defined as a heterogeneous group of clonal hematopoietic stem cell (HSC) malignant disorders which are characterized by bone marrow failure and dysplasia of blood cells. As continually terminated in AML, MDS are treated as the "pre-leukemia" condition. HERG K+ channels, being three subtypes: HERG1, HERG.2, HERG3, are expressed transiently at early stages of cells such as progenitor and stem cells, disappearing at later stages of cells like mature cells. Our previous work and others demonstrated that HERG1, as an oncoprotein, was over expressed in AML cells and played crucial roles in SDF-1induced leukemia cell migration. The expression and functional role of HERG1 K+ channels in MDS development is not reported. We investigated the HERG1 K+ channels expression and explored the functional link between HERG1 K+ channels and MDS progression Methods: The expression of HERG1 K+ channels in untreated MDS, AML patients and normal control was detected by flow cytometry. The roles of HERG1K+ channels in regulation of SKM-1 cell proliferation, apoptosis and cell cycle by CCK-8 assay and flow cytometry, respectively. Results: We observed that the expression of HERG1 K+ channels on bone marrow (BM) mononuclear cells (MNCs) in MDS patients was significantly higher than that in the controls (42 ± 7.62% vs 19.8 ± 2.79%, p < 0.01) , but was lower than that in AML (42 ± 7.62% vs 52.18 ± 9.72%, p < 0.01). MDS subtypes mainly contained refractory cytopenia unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blast I (RAEB-I), refractory anemia with excess blasts II (RAEB-II) and MDS-unclassified (MDS-U). We next analyzed percentage of HERG1 K+ channels in MDS subtypes and found that level of HERG1 K+ channels on the MNCs in each subtypes of MDS was significant higher than that in the control group (Control: 19.8 ± 2.79%, MDS-RCUD: 30.91 ± 1.48%, MDS-RCMD: 39.06 ± 2.47%, MDS-RAEB-I: 44.76 ± 5.54%, MDS-RAEB-II: 49.69 ± 3.28%, p < 0.01), suggesting that HERG1 K+ channels expression might be positively associated with malignancy degree of MDS. HSCs played important roles in the pathophysiology of MDS. Our results also revealed that with the increase of malignancy degree, the percentage of HERG1K+ channels on CD34+CD38- derived cells from MDS subtypes tended to elevate in corresponding MDS subtypes (MDS-RCUD: 61.21 ± 9.46%, MDS-RCMD: 65.66 ± 4.57%, MDS-RAEB-I: 72.35 ± 9.38%, MDS-RAEB-II: 75.71 ± 4.24%, p < 0.05), which supported the notion in other way that MDS are HSC malignant disorders. The over-expression of HERG1 K+ channels on CD34+CD38- cells in MDS patients might be correlated with the oncogenesis of MDS. In addition, the prognosis stratification of MDS patients was performed according to International Prognostic Scoring System (IPSS) scores and the untreated MDS cohort was categorized as four risk groups: Low-R, Int-1-R, Int-2-R and High-R. Our results showed that there was a positively correlation between HERG1 level and IPSS scores of patients (Low-R: 31.93 ± 3.47%, Int-1-R: 39.95 ± 5.76%, Int-2-R: 45.94 ± 6.34%, High-R: 49.05 ± 3.04%, p < 0.01). This showed that expression level of HERG1 K+ channels was helpful for predicting the prognosis of de novo MDS. Furthermore, we analyzed HERG1 K+ channel role on MDS cell proliferation and apoptosis. Incubation with 0, 10, 20 uM E-4031 ( HERG K+ channels inhibitor) with SKM-1 cells (MDS cell line) for 48 h, the results showed that blockage of HERG1 decreased the proliferation of SKM-1 cells but had rarely effects on cell apoptosis and cell cycle distribution. In consistent with other studies, HERG1 K+ channels had already been shown to be necessary for growth of cancer cells through specific activities independent of cell cycle such as interacting with TNFR1 protein which could activate NF-κB to facilitate cell proliferation and favoring transduction of growth signals by MAP kinase/c-fos pathway. These findings showed that physiological activity of HERG1 K+ channels was crucial for MDS cell proliferation and HERG1 K+ channels may be a potential therapeutic target for MDS. Conclusion: Briefly, our study firstly showed that HERG1 K+ channels were aberrantly over-expressed on MDS stem cells, mononuclear cells, and positively associated with malignancy degree of MDS. HERG1 K+ channels functionally contribute to MDS progression and may be a potential therapeutic target for MDS. Disclosures No relevant conflicts of interest to declare.

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Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Blood ◽

10.1182/blood.v116.21.4786.4786 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4786-4786

Author(s):

Fang Zheng ◽

Huiyu Li ◽

Fang Liu ◽

Wen Du ◽

Shiang Huang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Leukemic Cell ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

K Channels

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Human bone marrow mesenchymal stem cells suppress the proliferation of hepatic stellate cells by inhibiting the ubiquitination of p27

Biochemistry and Cell Biology ◽

10.1139/bcb-2017-0127 ◽

2017 ◽

Vol 95 (6) ◽

pp. 628-633 ◽

Cited By ~ 2

Author(s):

Liang Wang ◽

Guang Bai ◽

Fei Chen

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Cell Cycle Arrest ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Cycle Arrest ◽

Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) have considerable therapeutic potential for the treatment of end-stage liver disease. Previous studies have demonstrated that BMSCs secrete growth factors and cytokines that inactivate hepatic stellate cells (HSCs), which inhibited the progression of hepatic fibrosis. The aim of this study was to determine the mechanism by which BMSCs suppress the function of HSCs in fibrosis. Our results showed that co-culture of BMSCs and HSCs induced cell cycle arrest at the G10/G1 phase and cell apoptosis of HSCs, which finally inhibited the cell proliferation of HSCs. Consistent with the cell cycle arrest, co-culture of BMSCs and HSCs increased the abundance of the cell cycle protein p27. Mechanistically, we further uncovered that following the co-culture with BMSCs, the expression level of the E3 ligase S-phase kinase-associated protein 2 (SKP2) that is responsible for the ubiquitination of p27 was decreased, which attenuated the ubiquitination of p27 and increased the stability of p27 in HSCs. Collectively, our results indicated the potential involvement of the SKP2–p27 axis for the inhibitory effect of BSMCs on the cell proliferation of HSCs.

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Effect of rat bone marrow derived-mesenchymal stem cells on granulocyte differentiation of mononuclear cells as preclinical agent in cell-based therapy

Current Gene Therapy ◽

10.2174/1566523221666210519111933 ◽

2021 ◽

Vol 21 ◽

Author(s):

Ezzatollah Fathi ◽

Sheyda Azarbad ◽

Raheleh Farahzadi ◽

Sara Javanmardi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Growth Factors ◽

Mononuclear Cells ◽

Control Group ◽

Flow Cytometry Analysis ◽

Cell Lineages ◽

Experimental Group

Background: Bone marrow mononuclear cells (BM-MNCs), as a collection of hematopoietic and mesenchymal stem cells (MSCs), are capable of producing all blood cell lineages. The use of cytokines, growth factors, or cells capable of secreting these factors will help in stimulating the proliferation and differentiation of these cells into mature cell lines. On the other hand, MSCs are multipotent stromal cells that can be differentiated into various cell lineages. Moreover, these cells can control the process of hematopoiesis by secreting cytokines and growth factors. The present study aimed to investigate the effect of BM-derived MSCs on the differentiation of MNCs based on the assessment of cell surface markers by flow cytometry analysis. Methods: For this purpose, the MNCs were purified from rat BM using density gradient centrifugation. After that, they were cultured, expanded, and characterized. Next, BM-derivedMSCs were co-cultured with MNCs and then were either cultured with MNCs alone (control group) or co-cultured MNCs with BM derived-MSCs (experimental group). Finally, they were collected on day 7 and subjected to flow cytometry analysis for granulocyte markers and ERK protein’s investigation. Results: It was found that the expression levels of CD34, CD16, CD11b, and CD18 granulocyte markers, as well as protein expression of ERK, have significantly increased in the experimental group compared to the control group. Conclusion: Therefore, it can be concluded that MSCs could affect the granulocyte differentiation of MNCs via ERK protein expression, which is a key component of the ERK signaling pathway.

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Isolation of Human Bone Marrow Mesenchymal Stem Cells by a Novel Monoclonal Antibody, ZUC3.

Blood ◽

10.1182/blood.v108.11.2562.2562 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2562-2562

Author(s):

Xiaoyu Lai ◽

He Huang ◽

Li Huang ◽

Fenfang Zeng

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Sorting ◽

Mononuclear Cells ◽

Human Mscs ◽

Bone Marrow Mononuclear Cells ◽

Multiple Differentiation

Abstract Objective: Due to absence of a single definitive marker of mesenchymal stem cells (MSCs) and low incidence in human bone marrow, the primary culture of MSCs, conventionally isolated with its characteristic of adherent, were considered to be heterogeneous containing of several subpopulations, which had currently limited our understanding of their biology and therapeutic applications. In our previous study, a novel murine monoclonal antibody (McAb) ZUC3 was produced by hybridoma technology, which was specifically reactive with human MSCs, while showed negative cross-reactivity when screened against a variety of human tissues. Now, ZUC3 antigen positive MSCs population would be further identified by magnetic-activated cell sorting (MACS). Methods: Bone marrow were taken from the iliac crest of normal healthy adult volunteers, and mononuclear cells were separated by density gradient centrifugation, then separated into positively- and negatively-labelled fractions with McAb ZUC3 by immunomagnetic activated cell sorting. The purity of positive cells was analyzed by flow cytometry, then ZUC3 antigen positive and negative cells were plated respectively in human MSCs medium consisting of 10% FBS, LG-DMEM. Characteristics of ZUC3 antigen positive cells phenotype was analyzed by flow cytometry, and proliferation and multiple differentiation potential of the cells was observed in vitro. Results: Flow cytometric analysis showed that ZUC3 antigen expression by cultured MSCs and mononuclear cells derived from bone marrow were 91.31±2.92%, 0.96±0.28% respectively, and western blotting showed the molecular mass of antigen was about 33KD. The purity of the recovered fractions for ZUC3 by MACS was 76.82±6.32%. The positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. It was demonstrated that ZUC3 antigen positive cells continued growth with spindle-shape, extending beyond 30 population doublings in long-term culture. Analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for CD29, CD44, CD105, CD106, and lack typical hematopoietic antigens such as CD14, CD34, CD45, HLA-DR, which demonstrated that ZUC3 postive cells sorted from bone marrow mononuclear cells by McAb were MSCs. With proper medium, the ZUC3 antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, NSE and NF-M. Conclusion: ZUC3 McAb was a specific surface marker against human MSCs for cell sorting. The ZUC3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation.

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TSPAN1 promotes human pancreatic cancer cells proliferation by modulating CDK1 via Akt

10.1101/2020.05.29.123091 ◽

2020 ◽

Author(s):

Xin Wang ◽

Xiaozhuo Gao ◽

Jiaxun Tian ◽

Rui Zhang ◽

Yun Qiao ◽

...

Keyword(s):

Cell Cycle ◽

Pancreatic Cancer ◽

Cell Proliferation ◽

Therapeutic Target ◽

Human Pancreatic Cancer ◽

Potential Therapeutic Target ◽

Qrt Pcr ◽

Pancreatic Cancers ◽

Pancreatic Cancer Cells ◽

M Phase

AbstractBackgroundTo explore the potential therapeutic target to treat pancreatic cancers, Tspan1 was detected in human pancreatic cancer tissue and human pancreatic ductal adenocarcinoma cells and functional role of Tspan1 on proliferation was explored and the mechanism was investigated.Materials and MethodsTspan1 in PCC tissue and PDAC cell lines was measured by qRT-PCR and Western blot. Tspan1 was knock-downed and over-expressed in cells via transfection with Tspan1-siRNA and pLNCX-TSPAN1-cDNA, cell survival, proliferation and cell cycle were measured with MTT, Alamar blue and Flow Cytometry assay. The mRNA and protein expression were assessed by qRT-PCR and Western blotting. The expression of PI3K, Akt and p-Akt were detected, and CDK1 siRNA and specific inhibitor of Akt were used to explore the mechanism of TSPAN1 promoting PDAC cells proliferation.ResultsTspan1 expression in PCC tissue and PDAC cells was increased. Transfection of siRNA targeting Tspan1 in BxPC3 and PNAC-1 cells obviously decreased cell proliferation and down-regulated CDK1 expression. Consistently, both cell proliferation and CDK1 expression in BxPC3 and PNAC-1 cells were up-regulated with pLNCX-TSPAN1-cDNA transfection. Cell cycle analysis showed that after knockdown of Tspan1 the G2/M phase ratio was increased to cause mitosis arrest, and TSPAN1 overexpression caused cell cycle transition from G2 to M phase to promote cell proliferation. And these were dependent on the modulation of CDK1 expression via Akt.ConclusionTspan1 up-regulates CDK1 expression via activating Akt to promote human PCC cell proliferation and silencing of Tspan1 may be a potential therapeutic target to treat pancreatic cancers.

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Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow

Cell Proliferation ◽

10.1111/j.1365-2184.2007.00458.x ◽

2007 ◽

Vol 40 (5) ◽

pp. 656-670 ◽

Cited By ~ 55

Author(s):

X. L. Deng ◽

C. P. Lau ◽

K. Lai ◽

K. F. Cheung ◽

G. K. Lau ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Potassium Channels ◽

Cycle Dependent

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The Combination of AMD3100 Plus GROβ Rapidly Mobilizes Hematopoietic Stem Cells with Enhanced Homing, Adhesion and Survival Properties

Blood ◽

10.1182/blood.v112.11.71.71 ◽

2008 ◽

Vol 112 (11) ◽

pp. 71-71 ◽

Cited By ~ 1

Author(s):

Louis M. Pelus ◽

Pratibha Singh

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Mononuclear Cells ◽

Annexin V ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Donor Chimerism ◽

Stem And Progenitor Cells ◽

Blood Stem Cells

Abstract Mobilized peripheral blood stem cells (PBSC) are the primary cell source for hematopoietic transplantation. In mice, combined single administration of AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4 with GROβ, a CXCR2 agonist, rapidly mobilizes hematopoietic stem and progenitor cells (HSPC) within 15 minutes of administration that is equal or greater in magnitude to a multiday regimen of G-CSF and demonstrates significant synergy compared to either compound used alone. In non-competitive transplant models, AMD3100 plus GROβ mobilized 6.2±0.3 CRU/105 PBSC compared to 1.8±0.2, 1.9±0.2 and 2.0±0.3 CRU, respectively, for G-CSF, AMD3100 or GROβ used alone. Furthermore, AMD3100 plus GROβ mobilized long-term repopulating cells that show superior hematopoietic engrafting capacity in lethally irradiated mice. Analysis of chimerism at 6 months post competitive transplant of mobilized PBSC compared to normal bone marrow at a ratio of 1:1, showed 15±2%, 10±2% and 28±2% donor chimerism for G-CSF, AMD3100 and GROβ mobilized PBSC, respectively, whereas the equivalent number of AMD3100 plus GROβ mobilized PBSC produced 55±6% donor chimerism. In order to define the mechanism(s) responsible for increased engraftment of AMD3100 plus GROβ mobilized PBSC, we performed extensive phenotypic and functional analysis of combination AMD3100 plus GROβ mobilized HSPC and compared them to HSPC mobilized by G-CSF, AMD3100 or GROβ used alone. Flow cytometry analysis of highly enriched HSC using SLAM family receptor markers demonstrated that the AMD3100 plus GROβ mobilized PBSC population contained 1.8±0.1, 3.2±0.15 and 1.6±0.18 fold more CD150+ CD48− SKL cells, respectively, than the PBSC products mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, a higher proportion of AMD3100 plus GROβ mobilized CD150+ CD48− SKL cells expressed higher levels of the adhesion receptors CD11a, CD49e and CD49d. Cell cycle analysis demonstrated that AMD3100 plus GROβ mobilized PBSC contained more cells (92±4 %) in G0/G1 phase of the cell cycle than PBSC mobilized by G-CSF (82±6%), AMD3100 (76±4%) and GROβ (85±2%) alone. In homing studies using CFSE labeled mobilized mononuclear cells injected into lethally irradiated mice, ~2-fold more SKL cells mobilized by AMD3100 plus GROβ homed to bone-marrow compared to SKL cells mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, analysis of apoptosis in homed CFSE+ SKL cells indicated that 2.92±0.4% of homed SKL cells mobilized by AMD3100 plus GROβ were Annexin-V positive vs. 6.4±1.5%, 10.4±2.6% and 4.5±1.2%, respectively, for homed SKL cells mobilized by G-CSF, AMD3100 or GROβ alone, suggesting that mobilization by AMD3100 and GROβ in combination results in enhanced survival of mobilized HSC. Our results suggest that the enhanced engraftment observed upon transplantation of PBSC mobilized by combination treatment with AMD3100 plus GROβ in results from mobilization of a greater number of more primitive quiescent HSC that have enhanced intrinsic homing, adhesion and survival properties.

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Downregulation of THAP11 by Bcr-Abl Promotes c-Myc-Mediated CML Cell Proliferation.

Blood ◽

10.1182/blood.v114.22.3255.3255 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3255-3255

Author(s):

Daisuke Yokota ◽

Satoki Nakamura ◽

Tomonari Takemura ◽

Isao Hirano ◽

Shinya Fujisawa ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Cell Lines ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Abl Kinase ◽

Abl Kinase Inhibitors

Abstract Abstract 3255 Poster Board III-1 [Background and Purpose] THAP11 (thanatos-associated protein 11) is one of the zinc-dependent, sequence-specific DNA-binding factors, which regulate cell proliferation, apoptosis and cell cycle. It has been reported that THAP11 is ubiquitously expressed in normal tissues and frequently downregulated in several tumors. In CML cells, we found that THAP11 expression was inhibited. We investigated the mechanisms of the suppression of THAP11 and its function in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of THAP11 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the expression of c-Myc in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl siRNA, THAP11 siRNA, or THAP11 cDNA respectively. Moreover, THAP11 expression, c-Myc expression, and the colony counts of CFU-GEMM, CFU-GM, and BFU-E were analyzed in CML stem/progenitor cells transfected with Bcr-Abl siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfected with Bcr-Abl siRNA, the expressions of THAP11 and c-Myc mRNA and protein were significantly increased compared to untreated cells. On the other hand, in CML cells transfected with the THAP11 cDNA, the c-Myc expression was suppressed. The overexpression of THAP11 induced G1 cell cycle arrest through p27 and p21 accumulation, and inhibited the CML cell proliferation. Moreover, in CML stem/progenitor cells obtained from patients with CML, the transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of THAP11 mRNA and protein, and decreased the c-Myc expression and the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of THAP11, the depletion of THAP11 induced c-Myc expression, and induced the proliferation of CML cells through cell cycle progression. Moreover, induction of THAP11 expression inhibited the proliferation of CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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Effect of miR-124 on the Biological Characteristics of Bone Marrow Mesenchymal Stem Cells in Tumor Microenvironment

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2235 ◽

2020 ◽

Vol 10 (2) ◽

pp. 151-156

Author(s):

Linfu He ◽

XiaoyanHe ◽

Xiaocui Liu ◽

Wenjing Shi ◽

PeiXu ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Tumor Microenvironment ◽

Glioma Cells ◽

Biological Characteristics ◽

Brain Glioma ◽

Marrow Mesenchymal Stem Cells

miR-124 affects migration and differentiation of mesenchymal stem cells (MSCs), but its role in bone marrow mesenchymal stem cells (BMSCs) in tumor microenvironment remain unclear. Therefore, our study aims to assess miR-124’s role in BMSCs in glioma microenvironment. BMSCs were isolated and co-cultured with glioma cells using Transwell chamber and then transfected with miR-124 mimic/NC followed by analysis of BMSCs biological characteristics (cell proliferation by CCK8, miR-124 level by Real-time PCR, cell cycle and apoptosis by flow cytometry, and cell migration by Transwell). There were significant differences of cell proliferation, migration, apoptosis and cell cycle in BMSCs group compared to co-culture and co-culture+ miR-NC group (P < 0.05), without no difference compared to co-culture + miR-124 group. In addition, co-culture + miR-124 also showed significant differences of BMSCs' biological characteristics (P < 0.05). miR-124 can alter the biological characteristics of BMSCs in the tumor microenvironment simulated by C6 brain glioma cells.

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Enhancement of Immunoregulatory Function of Modified Bone Marrow Mesenchymal Stem Cells by Targeting SOCS1

BioMed Research International ◽

10.1155/2018/3530647 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10

Author(s):

Xiaoming Zhang ◽

Fei Hua ◽

Ziying Yang ◽

Yueqiu Chen ◽

Xiaomei Teng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Mononuclear Cells ◽

Treg Cells ◽

Control Group ◽

Mrna Levels ◽

Blank Control Group ◽

Marrow Mesenchymal Stem Cells

Objective. The study aim to investigate the role of microRNA-155 (miR-155) on the immunoregulatory function of bone marrow mesenchymal stem cells (MSCs). Methods. MSCs were isolated from 2-week-old Sprague-Dawley rats and identified by flow cytometry using anti-CD29, anti-CD44, anti-CD34, and anti-CD45 antibodies. MSCs were transfected with miR155-mimics, miR155-inhibitor, and control oligos, respectively, and then cocultured with spleen mononuclear cells (SMCs). The mRNA levels of Th1, Th2, Th17, and Treg cell-specific transcription factors (Tbx21, Gata3, Rorc, and Foxp3, resp.) and the miR-155 target gene SOCS1 were detected by quantitative real-time PCR (qPCR) in SMCs. The proportion of CD4+ FOXP3+ Treg cells was detected by flow cytometry. In addition, the effects of MSCs transfected with miR-155 on the migration of rat SMCs were investigated by transwell chamber. Results. CD29 and CD44 were expressed in MSCs, while CD34 and CD45 were negative. The percentage of CD4+ FOXP3+ Treg cells in the SMC population was significantly higher compared with that noted in SMCs control group (p<0.001) following 72 hours of coculture with miR155-mimics-transfected SMCs. In contrast, the percentage of CD4+ FOXP3+ Treg cells in the SMCs cocultured with miR155-inhibitor-transfected MSCs was significantly lower compared with that noted in SMCs control group (p<0.001). MiR155-mimics-transfected MSCs inhibited the expression of Tbx21, Rorc, and SOCS1, while the expression of Gata3 and Foxp3 was increased. In contrast to the downregulation of the aforementioned genes, miR155-inhibitor-transfected MSCs resulted in upregulation of Tbx21, Rorc, and SOCS1 expression levels and inhibition of Gata3 and Foxp3. In the transwell assay, miR155-mimics-transfected MSCs exhibited lower levels of SMCs migration, while the miR155-inhibitor-transfected MSCs demonstrated significantly higher levels of migration, compared with the blank control group (p<0.01, resp.). Conclusion. miR-155 favors the differentiation of T cells into Th2 and Treg cells in MSCs, while it inhibits the differentiation to Th1 and Th17 cells.

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