scholarly journals HIF2α Upregulates the Migration Factor ODZ1 under Hypoxia in Glioblastoma Stem Cells

2022 ◽  
Vol 23 (2) ◽  
pp. 741
Author(s):  
María Carcelén ◽  
Carlos Velásquez ◽  
Veronica Vidal ◽  
Olga Gutierrez ◽  
Jose L. Fernandez-Luna
Keyword(s):  
Stem Cells ◽  
Transcriptional Control ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Consensus Binding Site ◽  
Glioblastoma Stem Cells ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Luciferase Reporter Plasmid ◽  
Migration Factor

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

scholarly journals Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

2020 ◽  
Author(s):  
Yijing Chu ◽  
Chongyu Yue ◽  
Wei Peng ◽  
Weiping Chen ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chorionic Villus ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Upstream Regulator ◽  
Pathway Analyses ◽  
Stat3 Signalling

Abstract Objectives Trophoblast dysfunction during pregnancy is fundamentally involved in preeclampsia. The aim of this study was to understand how human chorionic villous mesenchymal stem cells (CV-MSCs) operate in regulation of trophoblast function.Materials and Methods We treated trophoblasts with CV-MSC supernatant under hypoxic conditions, and transcriptome and pathway analyses of trophoblasts were performed. Western blotting and PCR analysis were used to examine the JAK2, STAT3 and autophagy associated protein expression levels in trophoblasts.Results The CV-MSC supernatant treatment markedly enhanced proliferation, invasion and autophagy. The RNA-seq revealed JAK2/STAT3 signalling as an upstream regulator, and STAT3 mRNA and protein levels increased during CV-MSC treatment. Inhibition of JAK2/STAT3 signalling reduced autophagy, survival and invasion of trophoblasts even in the presence of CV-MSCs, and blocking autophagy did not affect STAT3 activation in trophoblasts treated with CV-MSCs. Importantly, overexpression of STAT3 increased the levels of autophagy in trophoblasts; thus, it regulated positively autophagy in hypoxic trophoblasts. Human placental explants also proved our finding, in which STAT3 was activated and LC3B-II levels were increased by CV-MSC treatment.Conclusions Our data suggest that CV-MSC-dependent activation of JAK2/STAT3 signalling is a prerequisite for upregulation of autophagy in trophoblasts.

scholarly journals MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Yicai Zhang ◽  
Yi Sun ◽  
Jinlong Liu ◽  
Yu Han ◽  
Jinglong Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

TPO-Induced miR-150 Down-Regulates c-Myb in Primary Megakaryocytes and a Megakaryocytic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1238-1238 ◽  
Author(s):  
Charlene F. Barroga ◽  
Hang Pham ◽  
Kenneth Kaushansky
Keyword(s):  
Transcription Factor ◽  
Western Blotting ◽  
Luciferase Activity ◽  
Mrna Translation ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Protein Levels ◽  
Micro Rnas

Abstract Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals Skeletal muscle interleukin-6 regulation in hyperthermia

2013 ◽  
Vol 305 (4) ◽  
pp. C406-C413 ◽  
Author(s):  
Steven S. Welc ◽  
Andrew R. Judge ◽  
Thomas L. Clanton
Keyword(s):  
Heat Shock ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
Heat Shock Factor 1 ◽  
Activator Protein 1 ◽  
Regulatory Pathways ◽  
Luciferase Reporter Plasmid

We previously reported that IL-6 production is acutely elevated in skeletal muscles exposed to ≥41°C, but the regulatory pathways are poorly understood. The present study characterizes the heat-induced transcriptional control of IL-6 in C2C12 muscle fibers. Hyperthermia exposure (42°C for 1 h) induced transcription from an IL-6 promoter-luciferase reporter plasmid. Heat shock factor-1 (HSF-1), a principal mediator of the heat shock response, was then tested for its role in IL-6 regulation. Overexpression of a constitutively active HSF-1 construct increased basal (37°C) promoter activity, whereas overexpression of a dominant negative HSF-1 reduced IL-6 promoter activity during basal and hyperthermia conditions. Since hyperthermia also induces stress-activated protein kinase (SAPK) signaling, we tested whether mutation of a transcription site downstream of SAPK, (i.e., activator protein-1, AP-1) influences IL-6 transcription in hyperthermia. The mutation had no effect on baseline reporter activity but completely inhibited heat-induced activity. We then tested whether pharmacologically induced states of protein stress, characteristic of cellular responses to hyperthermia and known to induce SAPKs and HSF-1, would induce IL-6 production in the absence of heat. The proteasome was inhibited with MG-132 in one set of experiments, and the unfolded protein response was stimulated with dithiothreitol, thapsigargin, tunicamycin, or castanospermine in other experiments. All treatments stimulated IL-6 protein secretion in the absence of hyperthermia. These studies demonstrate that IL-6 regulation in hyperthermia is directly controlled by HSF-1 and AP-1 signaling and that the IL-6 response in C2C12 myotubes is sensitive to categories of protein stress that reflect accumulation of damaged or unfolded proteins.

scholarly journals Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yijing Chu ◽  
Chengzhan Zhu ◽  
Chongyu Yue ◽  
Wei Peng ◽  
Weiping Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chorionic Villus ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Upstream Regulator ◽  
Before And After ◽  
Pathway Analyses ◽  
Stat3 Signalling ◽  
Proliferation And Invasion

Abstract Background Trophoblast dysfunction during pregnancy is fundamentally involved in preeclampsia. Several studies have revealed that human chorionic villous mesenchymal stem cells (CV-MSCs) could regulate trophoblasts function. Results To understand how human chorionic villous mesenchymal stem cells (CV-MSCs) regulate trophoblast function, we treated trophoblasts with CV-MSC supernatant under hypoxic conditions. Treatment markedly enhanced proliferation and invasion and augmented autophagy. Transcriptome and pathway analyses of trophoblasts before and after treatment revealed JAK2/STAT3 signalling as an upstream regulator. In addition, STAT3 mRNA and protein levels increased during CV-MSC treatment. Consistent with these findings, JAK2/STAT3 signalling inhibition reduced the autophagy, survival and invasion of trophoblasts, even in the presence of CV-MSCs, and blocking autophagy did not affect STAT3 activation in trophoblasts treated with CV-MSCs. Importantly, STAT3 overexpression increased autophagy levels in trophoblasts; thus, it positively regulated autophagy in hypoxic trophoblasts. Human placental explants also proved our findings by showing that STAT3 was activated and that LC3B-II levels were increased by CV-MSC treatment. Conclusion In summary, our data suggest that CV-MSC-dependent JAK2/STAT3 signalling activation is a prerequisite for autophagy upregulation in trophoblasts. Graphic abstract

Bioengineered microRNA-1291 effects on pancreatic cancer cell proliferation and xenograft tumor growth.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 307-307
Author(s):  
Mei-Juan Tu ◽  
Zhijian Duan ◽  
Qianyu Zhang ◽  
Jing-Xin Qiu ◽  
Frank J Gonzalez ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Therapeutic Potential ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay

307 Background: MicroRNAs (miR) have proved to be vital regulators in the control of tumor progression. Our recent studies have revealed miR-1291 is downregulated in patient pancreatic cancer (PC) specimens and re-introduction of miR-1291 suppresses tumorigenesis of PC cells. We have developed a novel ncRNA bioengineering technology to produce a miR-1291 prodrug. In this study, we aimed to assess the effectiveness of this miR-1291 prodrug as a monotherapy, as well as in combination with chemotherapy, for treatment of PC. Methods: Sensitivity of PC cells to miR-1291 prodrug alone, gemcitabine plus nab-paclitaxel (Gem-nP) alone, and their combination was evaluated by CellTiter-Glo assay. Mature miR-1291 and ARID3B mRNA levels were determined by quantitative real-time PCR (q-PCR) assay. A luciferase reporter gene assay was used to validate interaction between miR-1291 and ARID3B 3’UTR. Target protein expression was examined by Western blot and immunofluorescence analyses. PANC-1 and PC patient-derived xenograft (PDX) mouse models were established and used to assess anti-tumor effects of miR-1291 monotherapy and combination therapy with Gem-nP. Results: Cytotoxicity assays showed that miR-1291 prodrug enhanced the sensitivity of PANC-1 and AsPC-1 cells to Gem-nP. Luciferase assays confirmed ARID3B as a target for miR-1291 as predicted by computational analysis. qPCR analysis demonstrated that miR-1291 prodrug was readily processed to mature miR-1291 and subsequently upregulated ARID3B mRNA levels. miR-1291 prodrug also elevated the protein levels of ARID3B. Co-administration of miR-1291 prodrug and Gem-nP increased caspase-3/7 and γH2AX levels in PC cells, compared to miR-1291 or Gem-nP treatment alone. In addition, systemic administration of in vivo-jet PEI formulated miR-1291 prodrug suppressed tumor growth in both a PANC-1 xenograft model and three PDX models, and largely enhanced the efficacy of Gem-nP. All treatments were well tolerated in mice in vivo. Conclusions: Our bioengineered miR-1291 prodrug has therapeutic potential as a monotherapy but also can act as a sensitizing agent to chemotherapy. This novel treatment approach should be further explored for PC.

The c-myb Protooncogene and microRNA (miR)-15a Form an Autoregulatory Loop That May Play a Role in Normal Human Erythropoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2642-2642
Author(s):  
Huiwu Zhao ◽  
Anna Kalota ◽  
Shenghao Jin ◽  
Alan Gewirtz
Keyword(s):  
Binding Sites ◽  
Luciferase Activity ◽  
Hek293t Cell ◽  
K562 Cells ◽  
Hematologic Malignancies ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Protein Levels ◽  
Normal Human ◽  
Myb Protein

Abstract The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor that contributes to lineage commitment, proliferation, and differentiation. Factors which regulate c-myb expression are of interest but remain incompletely defined. MicroRNAs (miRNAs) are being increasingly recognized as important regulators of cell development, and abnormalities in miRNA activity may also contribute to the pathogenesis of several hematologic malignancies. We speculated that miRNAs might also regulate c-Myb expression, a gene often aberrantly expressed in leukemia, lymphoma, and myeloma. Accordingly, we searched for potential miRNA binding sites in the 3′-UTR of the c-myb mRNA using the TargetScanS Target Database and identified 14 candidate miRNAs. Based on binding probability, three miRNAs (miR-15a, −107 and −150) were selected for further analysis using a luciferase reporter assay. 1191 bp of the human c-Myb 3′-UTR was subcloned downstream of the f-luc open reading frame to create the reporter construct (pBub1/Myb3U). This was co-transfected into HEK293T cell line with pRL-CMV (to normalize for transfection differences) and then either a control RNA oligonucleotide (ON) (miR-Control), miR-15a, miR-107, or miR-150 ONs. Relative luciferase activity of the pBub1/Myb3U construct was markedly diminished in cells co-transfected with miR-15a (74.1 ± 1.5%) or miR-107 (68.2 ± 5.3%) ONs, but only modestly with miR-150 ON (22.1 ± 4.2%). Functionality of miR-15a site was further tested by mutating the predicted miR-15a binding sites. This resulted in a 2 to 3 fold increase in luciferase activity, suggesting that miR-15a bound the predicated sites, and that they might be physiologically relevant. To test this possibility, miR-15a ONs were transfected into K562 human myeloid leukemia cells and the effects on c-Myb mRNA and MYB protein levels were determined. As expected with functional miRNA, c-myb mRNA levels did not change when compared to control treated cells, as measured by quantitative real-time PCR, but Myb protein levels were significantly decreased. Additionally, when analyzed by flow cytometry, miR-15a transfected cells were found to be arrested in G1 as might be expected in a Myb knockdown experiment. Conversely, when K562 cells were transfected with a methylphosphonate inhibitor (antisense) of miR-15a, endogenous c-myb expression increased. Moreover, exogenous expression in K562 cells of a c-Myb mRNA construct devoid of its 3′-UTR partially rescued the miR-15a induced cell cycle arrest. Interestingly, miR-15a levels were found to vary inversely with c-Myb mRNA expression levels in normal human CD34+ cells stimulated to develop along the erythroid, but not the myeloid lineage. These results suggested that miR-15a might play a specific, and potentially important role in regulating normal human erythropoiesis by modulating the expression of c-Myb, though we cannot exclude the possibility that additional miR-15a targets are also important. Finally, a siRNA mediated knockdown of c-Myb expression in K562 cells resulted in reduction of miR-15a expression, suggesting the possibility that Myb and miR-15a autoregulate their expression through a negative feed-back loop. We conclude that miR-15a regulates c-Myb expression in a physiologically significant, lineage specific manner in normal human hematopoietic cells. Since miR-15a localizes to ch13q14, a region often deleted in hematologic malignancies, we postulate that it might also play a role in leukemogenesis. This possibility is under active investigation in our laboratory.

Analysis of Wnt and Hedgehog Pathways Regulating Protein Kinases CK1 and CK2 in Acute Myeloid Leukemia Cells and Stem Cells: Correlation with the Expression of Wnt and Hedgehog Targets and Biological and Clinical Features.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2501-2501
Author(s):  
Alessandra Brancalion ◽  
Laura Quotti Tubi ◽  
Sabrina Manni ◽  
Anna Cabrelle ◽  
Fortunato Zaffino ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Protein Kinases ◽  
Myeloid Leukemia ◽  
Mrna Levels ◽  
Expression Data ◽  
Protein Levels ◽  
Chemical Inhibitors ◽  
Hh Signaling ◽  
Acute Myeloid

Abstract Abstract 2501 Background. Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/βcatenin and the Hedgehog (Hh) signaling pathways regulate the balance between self-renewal and commitment of both normal hematopoietic stem cells (HSC) and LSC. Serine-Threonine protein kinases CK1 and CK2 phosphorylate members of Wnt and Hh and are therefore potential regulators of HSC and LSC biology; however, their function in HSC and LSC is unknown. Purpose. In the present work we have investigated the mRNA and protein levels of CK1 and CK2 in acute myeloid leukemia (AML) blasts and LSC isolated from patients. We have also integrated the expression data of CK1 and CK2 with the expression data of Wnt and Hh signaling pathways gene targets and with biological and clinical parameters of a group of AML patients and cell lines. We also tested the effects on the growth of AML blasts of CK1 and CK2 small chemical inhibitors. The aim of this study was to gain insights into the function of these pivotal protein kinases and on their expression levels relative to biological, clinical and prognostic variables that dictate the outcome of AML patients. Methods. mRNA was extracted from HSC and FACS-sorted LSC (as defined as Lineage- CD34+ CD38- CD123+ CD90+); proteins were obtained from the bulk of blasts from fifteen AML patients. Quantitative RT-PCR was employed to assess mRNA levels of: CK1α, CK1γ, CK2α (catalytic) and CK2β (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1α, CK2α, CK2β, p53, phospho-Ser529 NF-κB and phospho-Ser13 Cdc37 (two direct CK2 target sites) protein levels. Normal CD34+ bone marrow cells were used as controls. CK2 chemical inhibitors (CX4945 and tTBB) were assayed on LSC and AML cells. Results. CK1α, CK2α and CK2β mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1γ mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2β but high levels of CK2α, indicating a possible CK2β-independent function of CK2α. FoxO1, FoxO3 mRNAs were found upregulated while Lef1 and CyclinD1 ones were found slightly and Axin2 one strongly diminished, respectively. Ptch, Smo, Gli1, Gli2 and Gli3 mRNAs were unchanged or reduced, while we observed a strong upregulation of that of Bmi1. A correlation between the overexpression of CK1α and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2α high-expressing cases, TP53 was found downregulated, while phospho-Ser529 p65 upregulated. Importantly, inhibition of CK2 with selective compounds caused AML cell and LSC growth arrest, a restoration of TP53 and a downregulation of phospho-Ser529-NF-kB p65 and phospho-Ser13 Cdc37 protein levels. Conclusions. The present study is the first to report on the expression of CK1 and CK2 kinases in normal HSC and LSC. CK1 (α) and CK2 (α and β) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1γ mRNA levels were low. Wnt and Hh pathways genes were differentially upregulated pointing to a gene specific LSC-associated function. Moreover, our data with CK2 inhibitors suggest that CK2 could be a suitable therapeutic target to eradicate residual AML LSCs. Future research will assess more in depth the in vivo functional role of CK1 and CK2 in HSC and LSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Let-7c regulates proliferation and osteodifferentiation of human adipose-derived mesenchymal stem cells under oxidative stress by targeting SCD-1

2019 ◽  
Vol 316 (1) ◽  
pp. C57-C69 ◽  
Author(s):  
Zihui Zhou ◽  
Yuanshan Lu ◽  
Yao Wang ◽  
Lin Du ◽  
Yunpeng Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Postmenopausal Osteoporosis ◽  
Luciferase Reporter ◽  
Matrix Mineralization ◽  
Protein Levels ◽  
Micro Rnas

Osteoporosis is a progressive bone disease characterized by decreased bone mass and density, which usually parallels a reduced antioxidative capacity and increased reactive oxygen species formation. Adipose-derived mesenchymal stem cells (ADMSCs), a population of self-renewing multipotent cells, are a well-recognized source of potential bone precursors with significant clinical potential for tissue regeneration. We previously showed that overexpressing stearoyl-CoA desaturase 1 (SCD-1) promotes osteogenic differentiation of mesenchymal stem cells. Micro-RNAs (miRNAs) are noncoding RNAs recently recognized to play key roles in many developmental processes, and miRNA let-7c is downregulated during osteoinduction. We found that let-7c was upregulated in the serum of patients with postmenopausal osteoporosis compared with healthy controls. Levels of let-7c during osteogenic differentiation of ADMSCs were examined under oxidative stress in vitro and found to be upregulated. Overexpression of let-7c inhibited osteogenic differentiation, whereas inhibition of let-7c function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase activity, and matrix mineralization. The luciferase reporter assay was used to validate SCD-1 as a target of let-7c. Further experiments showed that silencing of SCD-1 significantly attenuated the effect of let-7c inhibitor on osteoblast markers, providing strong evidence that let-7c modulates osteogenic differentiation by targeting SCD-1. Inhibition of let-7c promoted the translocation of β-catenin into nuclei, thus activating Wnt/β-catenin signaling. Collectively, these data suggest that let-7c is induced under oxidative stress conditions and in osteoporosis, reducing SCD-1 protein levels, switching off Wnt/β-catenin signaling, and inhibiting osteogenic differentiation. Thus, let-7c may be a potential therapeutic target in the treatment of osteoporosis and especially postmenopausal osteoporosis.

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