Inhibitory Effect of RNAi on Expression of bcr/abl Oncogene in K562 Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4850-4850
Author(s):  
Chun Wang ◽  
Xiaoxia Ma ◽  
Ju Wei ◽  
Youwen Qin ◽  
Shike Yan
Keyword(s):  
Cell Growth ◽  
Transfection Efficiency ◽  
Mrna Level ◽  
Flow Cytometric Analysis ◽  
K562 Cells ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
Obvious Effect ◽  
Western Blots ◽  
Oncogene Expression

Abstract The translocation t(9;22) can be found in 95% patients with chronic myeloid leukemia (CML). The resulting hybrid gene bcr/abl codes for a fusion protein with tyrosine kinase activity which activates signal transduction pathways, leading to uncontrolled cell growth. A promising anti-gene technology-RNA interference (RNAi) reported in recent years can disrupts the expression of the targeted cellular gene in a complicated manner in a variety of organisms and cell types. To inhibit CML bcr/abl oncogene expression with RNAi, we used chemically synthesized anti-bcr/abl small interfering RNAs( siRNAs), mismatch control siRNAs having the three point mutations to transfect the K562 cells for different time through the electroporation. EGFP plasmid was used as a positive control and the amount of fluorescently stained cells was determined by FCM. Inhibitory effects of siRNAs were demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by means of MTT assay and apoptosis was determined by Annexin V-FITC assay. The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. The anti-bcr/abl siRNAs reduced the bcr/abl mRNA level by 67%–72% without an obvious effect on abl mRNA levels. The K562 cells electroporated with anti-bcr/abl siRNAs contained less BCR/ABL protein than cells electroporated with mismatch control siRNAs or without any siRNAs. P210 was reduced to a very low level in Western blots, whereas the wild-type ABL protein was not influenced by the anti-bcr/abl siRNAs. Depletion of bcr/abl leading to increased apoptosis and reduction of cell viability. The flow cytometric analysis showed that the percentage of dead and apoptosic cells induced by anti-bcr/abl siRNAs for 24hrs and 48hrs were 42.10% and 53.33%, respectively, including 15.05% and 19.47% of the preliminary apoptosis cells, respectively. A significant induction of apoptosis was observed after transfection compared with untreated control(1.00%)and the mismatch control siRNAs (2.98%) (p<0.05). Anti-bcr/abl siRNAs inhibited cell growth of the K562 cells and the inhibitory rate was 47% and 56%, 24h and 48h after transfection respectively, whereas the mismatch control siRNAs had no such effect on K562 cells. At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study. Therefore, siRNAs may be potent tools for gene-specific modality against CML.

scholarly journals XCHD Inhibits C6 Cell Growth Primarily via the p53/Caspase Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Huiling Yu ◽  
Qinnuan Sun ◽  
Fen Yun ◽  
Xiaoyan Xu ◽  
Feng Ding ◽  
...  
Keyword(s):  
Cell Growth ◽  
Caspase 3 ◽  
Mrna Level ◽  
Time Effect ◽  
Caspase 8 ◽  
C6 Cells ◽  
Western Blots ◽  
Factors Associated ◽  
Caspase Pathway

The effects of XCHD on the proliferation of C6 cells and on factors associated with the microRNA-34a (miR-34a)/p53/caspase-3 signaling pathway in vitro were investigated. Methods. XCHD was purchased too much to complete the study. CCK-8 assay was used to measure the XCHD concentration, and qPCR was used to quantify miR-34a expression at the mRNA level. Apoptosis was assessed using TUNEL. Western blots were used to determine the p53, caspase-3, caspase-8, and Bcl-2 expression levels. Results. The optimal XCHD concentration and time effect for C6 cells were observed after 36 h of exposure to a concentration of 100 µg/ml XCHD. miR-34a expression increased 8 and 12 h after the addition of XCHD. The presence of XCHD decreased Bcl-2 expression but increased p53, cleaved caspase-3, Bax, and caspase-8 expression. When p53 was inhibited, miR-34a expression was unaffected by the addition of XCHD, Bcl-2 expression was low, and cleaved caspase-3, Bax, and caspase-8 expression increased. The inhibition of p53 promoted C6 cell growth when compared with C6 cells exposed to XCHD and with no inhibition of p53. Conclusions. XCHD inhibits C6 cell growth which was influenced by the p53/caspase pathway.

scholarly journals MicroRNA-130a–mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors

Blood ◽  
2011 ◽  
Vol 118 (25) ◽  
pp. 6649-6659 ◽  
Author(s):  
Mattias Häger ◽  
Corinna Cavan Pedersen ◽  
Maria Torp Larsen ◽  
Mette Klarskov Andersen ◽  
Christoffer Hother ◽  
...  
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Mrna Level ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Smad4 Protein ◽  
Tgf Β1

Abstract Smad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3′-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1–induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia–derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1–mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.

scholarly journals Improvement Activity of 1-Deoxynojirimycin in the Growth of Dairy Goat Primary Mammary Epithelial Cell through Upregulating LEF-1 Expression

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Shengyue Ji ◽  
Ming Liu ◽  
Yuping Zhang ◽  
Hongfu Zhang
Keyword(s):  
Mammary Gland ◽  
Cell Growth ◽  
Epithelial Cell ◽  
Growth And Development ◽  
Mrna Level ◽  
Dairy Goat ◽  
Breast Epithelial Cell ◽  
Mrna Levels ◽  
Dairy Goats ◽  
Breast Epithelial

LEF-1/wnt10b is one of the most important signaling pathways regulating mammary gland growth and development and is also a potential target for molecular breeding. In this work, 1-deoxynojirimycin (DNJ), a natural alkaloid extracted from plant mulberry or microorganism, was found to have a positive activity in primary breast epithelial cell growth of dairy goats. The findings showed that, compared to the control, 6 μM DNJ in the DMEM/F12 medium in vitro greatly improved the density of dairy goat breast epithelial cell and significantly increased the LEF-1 mRNA level (P<0.01) and thus enhanced cell growth. In addition, DNJ displayed a similar function in alleviating the growth suppression of epithelial cell and the decrease of LEF-1 mRNA level resulting from lentiviral-mediated LEF-1 knockdown. Simultaneously, no significant change of the mRNA levels of IGF-1 and Fgf10, the other two key regulators in mammary gland growth and development, could be detected. Furthermore, the mammary duct of DNJ-fed mouse illustrated a better development accompanied with a higher LET-1 mRNA level than that of the control. In conclusion, DNJ could improve breast epithelial cell growth through upregulating LEF-1 expression, which supplied a new means in studying mammary gland growth and development.

Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus

2017 ◽  
Vol 117 (03) ◽  
pp. 529-542 ◽  
Author(s):  
Zsolt Fejes ◽  
Szilárd Póliska ◽  
Zsolt Czimmerer ◽  
Miklós Káplár ◽  
András Penyige ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Platelet Activation ◽  
Mrna Level ◽  
K562 Cells ◽  
Mrna Levels ◽  
Supplementary Material ◽  
Calpain Inhibition

SummaryMegakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Diminished expression of ICOS, GITR and CTLA-4 at the mRNA level in T regulatory cells of children with newly diagnosed type 1 diabetes.

2009 ◽  
Vol 56 (2) ◽  
Author(s):  
Włodzimierz Łuczyński ◽  
Natalia Wawrusiewicz-Kurylonek ◽  
Anna Stasiak-Barmuta ◽  
Remigiusz Urban ◽  
Elzbieta Iłendo ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
T Regulatory Cells ◽  
Mrna Level ◽  
Flow Cytometric Analysis ◽  
Treg Cells ◽  
Future Research ◽  
Mrna Levels ◽  
Newly Diagnosed ◽  
Regulatory Cells

Diabetes mellitus is one of the most common chronic diseases in children. T regulatory cells (Tregs) modulate response to autoantigens and probably play a role in pathogenesis of type 1 diabetes (T1DM). The aim of the present study was the assessment of T regulatory cells including their percentages and expression of critical genes in these cells in children with newly diagnosed type 1 diabetes. The examined group consisted of 50 children with T1DM. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD4, anti-CD25 and anti-CD127 (=IL-7R). Additionally, T regulatory cells were isolated for assessment of mRNA levels for chosen genes with the real-time RT-PCR technique. The percentages of CD4(+)CD25(high)CD127(dim/-) were very low and did not differ between T1DM and control children. We did not observe any statistically significant differences between healthy and diabetic children in mRNA expression for FoxP3, IL-7R (CD127), IL-8RA, IL-10RA, IL-12A, IL-2RA (CD25), IL-21, STAT1, STAT3, SOCS2, SOCS3, TGF-beta1-R1, TGF-beta-R2 and TBX-21 genes. Interestingly the mRNA level for CTLA-4, ICOS1, IL-23, IL-27, SMAD3 and GITR were lower in Treg cells of children with diabetes compared to the control patients. No disturbances in the percentages of T regulatory cells in patients with diabetes but diminished expression of some elements important in Treg function could be the result of an immunologic imbalance accompanying the onset of the diabetes. The results of our study should be used in future research in the field of immunotherapy in pediatric diabetes.

scholarly journals Candida glabrata Med3 Plays a Role in Altering Cell Size and Budding Index To Coordinate Cell Growth

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Hui Liu ◽  
Lulin Kong ◽  
Yanli Qi ◽  
Xiulai Chen ◽  
Liming Liu
Keyword(s):  
Cell Growth ◽  
Cell Size ◽  
Candida Glabrata ◽  
Mrna Level ◽  
Growth Defect ◽  
Mrna Levels ◽  
Acetyl Coa ◽  
Content Type ◽  
Quantitative Expression ◽  
Transcriptomics Data

ABSTRACT Candida glabrata is a promising microorganism for the production of organic acids. Here, we report deletion and quantitative-expression approaches to elucidate the role of C. glabrata Med3AB (CgMed3AB), a subunit of the mediator transcriptional coactivator, in regulating cell growth. Deletion of CgMed3AB caused an 8.6% decrease in final biomass based on growth curve plots and 10.5% lower cell viability. Based on transcriptomics data, the reason for this growth defect was attributable to changes in expression of genes involved in pyruvate and acetyl-coenzyme A (CoA)-related metabolism in a Cgmed3abΔ strain. Furthermore, the mRNA level of acetyl-CoA synthetase was downregulated after deleting Cgmed3ab, resulting in 22.8% and 21% lower activity of acetyl-CoA synthetase and cellular acetyl-CoA, respectively. Additionally, the mRNA level of CgCln3, whose expression depends on acetyl-CoA, was 34% lower in this strain. As a consequence, the cell size and budding index in the Cgmed3abΔ strain were both reduced. Conversely, overexpression of Cgmed3ab led to 16.8% more acetyl-CoA and 120% higher CgCln3 mRNA levels, as well as 19.1% larger cell size and a 13.3% higher budding index than in wild-type cells. Taken together, these results suggest that CgMed3AB regulates cell growth in C. glabrata by coordinating homeostasis between cellular acetyl-CoA and CgCln3. IMPORTANCE This study demonstrates that CgMed3AB can regulate cell growth in C. glabrata by coordinating the homeostasis of cellular acetyl-CoA metabolism and the cell cycle cyclin CgCln3. Specifically, we report that CgMed3AB regulates the cellular acetyl-CoA level, which induces the transcription of Cgcln3, finally resulting in alterations to the cell size and budding index. In conclusion, we report that CgMed3AB functions as a wheel responsible for driving cellular acetyl-CoA metabolism, indirectly inducing the transcription of Cgcln3 and coordinating cell growth. We propose that Mediator subunits may represent a vital regulatory target modulating cell growth in C. glabrata.

scholarly journals Cell-Cycle–Dependent Regulation of Erythropoietin Receptor Gene

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1182-1188 ◽  
Author(s):  
Norio Komatsu ◽  
Keita Kirito ◽  
Yoshifumi Kashii ◽  
Yusuke Furukawa ◽  
Jiro Kikuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Growth ◽  
Receptor Gene ◽  
Mrna Level ◽  
State Level ◽  
Erythropoietin Receptor ◽  
Cycle Phase ◽  
Mrna Levels ◽  
Mobility Shift

Abstract To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF ), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

scholarly journals Absence of Calreticulin Phenocopies Cellular Abnormalities Induced By Calreticulin Exon-9 Mutation in Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1780-1780
Author(s):  
Laura Calabresi ◽  
Manjola Balliu ◽  
Niccolò Bartalucci ◽  
Simona Salati ◽  
Laura Maggi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloproliferative Neoplasms ◽  
Flow Cytometric Analysis ◽  
K562 Cells ◽  
Mrna Levels ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Exon 9 ◽  
T1 And T2 ◽  
In Cells

Abstract INTRODUCTION. Calreticulin (CALR) is mutated in 20% of pts with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most frequent mutations are a 52 bp deletion (T1) and a 5 bp insertion (T2) in exon 9, that cause a recurrent frameshift resulting in novel C-terminal sequence common to all mutant CALR proteins. Recent data indicate that interaction of mutant CALR with the thrombopoietin receptor MPL contributes to the abnormal megakaryocytopoiesis (Mk) of ET/PMF (Araki M et al & Chachoua I et al, Blood 2016; Elif S et al, Blood 2018), however full characterization of mechanisms pertaining to mutant CALR remains to be pursued. METHODS. By using CRISPR/Cas9 editing, we generated CALR knock-out (KO) variants starting from cord blood (CB) CD34+ cells, K562, UT7 and HL-60 cell lines, and CALR T1 variants from K562 and UT7 cells. Stable expression of CALR wild-type (WT), T1 and T2 was obtained by viral transfection of K562-KO cells. RESULTS. In the different genome-edited cell models, KO or T1 mutation did not result in appreciable changes in proliferation rate, cell cycle and apoptosis under standard culture conditions. However, UT7 KO and T1 cells were able to grow in the absence of GM-CSF, that was otherwise necessary for maintenance of WT counterpart, indicating cytokine independence similarly imparted by deletion or T1 mutation of CALR. To evaluate impact of mutated CALR on Mk commitment, we induced parental, KO and T1 K562 cells with phorbol-myristate acetate (PMA); at day 3 after induction, 60% and 48% of KO and T1 cells, respectively, expressed CD41/CD61 compared to 24% of parental cells (p<0.01). Similar findings were noted in KO K562 cells that were transfected with vectors encoding the WT, T1 and T2 mutated CALR: CD41/CD61+ cells increased from 30% in parental cells to 70%, 55%, 62%, respectively, in KO cells and cells expressing T1 and T2 mutant CALR (p<0.01). We also found that a greater proportion of KO and T1 UT7 cells, maintained in either GM-CSF and TPO, spontaneously expressed CD41/CD61 at d7 of culture (50-51% and 41-49%, respectively, for KO and T1 cells with GM-CSF or TPO compared to 26-25% of parental cells; p<0.01) and underwent morphologically recognizable Mk maturation. Mk clonogenic assays were initiated in plasma clot cultures by plating CB-derived CD34+ cells with CRISPR/Cas9-induced deletion of CALR (KO cells). KO CD34+ cells generated a number of CFU-Mk colonies that was 10-fold higher than untouched CD34+ cells (p<0.05). Overall, these data suggested that deletion of CALR results in promotion of Mk differentiation in cell lines and primary cells thereby mimicking the effects of CALR T1. Array phosphoproteomic assay in K562 KO cells showed phosphorylation levels of p38γ, p70S6k mTOR, Erk1 and Erk2 from 0.5 to >2-fold higher compared to parental cells. Then, we induced KO and T1 K562 cells to Mk differentiation in the presence of JAK2, mTOR and Erk1/2 -inhibitors; only mTOR and Erk1/2 -inhibitors significantly (P< 0.05) reduced Mk differentiation of T1 and KO cells by respectively 5- and 4- fold with mTOR inhibitor and 5- and 4.5- fold with Erk1/2 inhibitor compared to parental cells, that were largely insensitive to inhibitors (Figure). Isolated megakaryocytes from CALR-mutant patients were found to present abnormally increased release of Ca2+ from endoplasmic reticulum (ER) compared to control cells (Pietra D et al. Leukemia, 2016). We similarly found that both KO and T1 K562 cells presented impaired capability in restoring calcium homeostasis compared to parental cells under treatment with ionophores thapsigargin and ionomycin. Finally, we comparatively assessed MPO expression by flow cytometric analysis in parental and CALR KO HL-60 cell lines; increased myeloperoxidase (MPO) degradation was previously reported in MPN pts expressing CALR mutation (Theocharides A et al., Blood, 2016). We found significant reduction of MPO levels (9-fold lower, p<0.01) in KO compared to parental cells, while MPO mRNA levels were not affected by CALR KO, suggesting similarly enhanced degradation of MPO in cells with targeted deletion of CALR. CONCLUSIONS. Overall, our data suggest that the novel C-terminus of CALR originating from exon 9 mutations loses some physiologically functions that are phenocopied by absence of CALR protein in cells made KO for the gene in a MPL-independent context. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

2018 ◽  
Vol 18 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Maristella Bergamo dos Reis ◽  
Vanessa Silva Silveira ◽  
Regia Caroline Peixoto Lira ◽  
Carlos Gilberto Carlotti Jr ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Relevant Information ◽  
Protein Stabilization ◽  
Mrna Levels ◽  
Medulloblastoma Cell Line ◽  
Pediatric Medulloblastoma ◽  
Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

scholarly journals Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jianjun Jiang ◽  
Yining Shi ◽  
Jiyu Cao ◽  
Youjin Lu ◽  
Gengyun Sun ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Nlrp3 Inflammasome ◽  
Mrna Level ◽  
Scavenger Receptor ◽  
Nuclear Factor Κb ◽  
Inflammasome Activation ◽  
Mrna Levels ◽  
Irreversible Inhibitor ◽  
Nlrp3 Inflammasome Activation

Abstract Background This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. Methods Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1β and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. Results Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. Conclusion This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

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