The effect of antenatal depression and antidepressant treatment on placental tissue: a protein-validated gene expression study

Abstract Background Antenatal depression affects 10–20% of pregnant women. Around 2–4% of European pregnant women use antidepressant treatment, most commonly selective serotonin reuptake inhibitors (SSRIs). Poor pregnancy outcomes, such as preterm birth and low birth weight, have been described in women with antenatal depression and in pregnant women on SSRI treatment. However, the effects of antenatal depression and antidepressant treatment on the placenta are largely unknown. The aim of this work was to compare placental gene and protein expression in healthy women, women with untreated antenatal depression and women on antidepressant treatment during pregnancy. Methods Placental samples from 47 controls, 25 depressed and 45 SSRI-treated women were analysed by means of qPCR using custom-designed TaqMan low-density arrays (TLDAs) for 44 genes previously known to be involved in the pathophysiology of depression, and expressed in the placenta. Moreover, placental protein expression was determined by means of immunohistochemistry in 37 healthy controls, 13 women with untreated depression and 21 women on antidepressant treatment. Statistical comparisons between groups were performed by one-way ANOVA or the Kruskal–Wallis test. Results Nominally significant findings were noted for HTR1A and NPY2R, where women with untreated depression displayed higher gene expression than healthy controls (p < 0.05), whereas women on antidepressant treatment had similar expression as healthy controls. The protein expression analyses revealed higher expression of HTR1A in placentas from women on antidepressant treatment, than in placentas from healthy controls (p < 0.05). Conclusion The differentially expressed HTR1A, both at the gene and the protein level that was revealed in this study, suggests the involvement of HTR1A in the effect of antenatal depression on biological mechanisms in the placenta. More research is needed to elucidate the role of depression and antidepressant treatment on the placenta, and, further, the effect on the fetus.

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Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

Arthritis Research & Therapy ◽

10.1186/s13075-021-02504-z ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Matthew Mannarino ◽

Hosni Cherif ◽

Li Li ◽

Kai Sheng ◽

Oded Rabau ◽

...

Keyword(s):

Gene Expression ◽

Back Pain ◽

Protein Expression ◽

Disc Degeneration ◽

Cell Senescence ◽

Enzyme Linked Immunosorbent Assay ◽

Matrix Synthesis ◽

Gene And Protein Expression ◽

Pain Patients ◽

In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

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83 Spatially resolved transcriptomic and proteomic investigation of breast cancer and its immune microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.083 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A91-A91

Author(s):

Jennifer Chew ◽

Cedric Uytingco ◽

Rapolas Spalinskas ◽

Yifeng Yin ◽

Joe Shuga ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Tumor Microenvironment ◽

Protein Expression ◽

Protein Detection ◽

Response To Therapy ◽

Pathological Examination ◽

Multi Drug Resistance ◽

Spatially Resolved ◽

Gene And Protein Expression

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous extracellular structures and cell types such as endothelial cells, immune cells, and fibroblasts that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in cancer development and progression and can significantly affect a tumor’s response to therapy and capacity for multi-drug resistance. High resolution analyses of gene and protein expression with spatial context can provide deeper insights into the interactions between tumor cells and surrounding cells within the TME, where a better understanding of the underlying biology can improve treatment efficacy and patient outcomes. Here, we demonstrated the ability to perform streamlined multi-omic tumor analyses by utilizing the 10X Genomics Visium Spatial Gene Expression Solution for FFPE with multiplex protein enablement. This technique simultaneously assesses gene and protein expression to elucidate the immunological profile and microenvironment of different breast cancer samples in conjunction with standard pathological methods.MethodsSerial (5 µm) sections of FFPE human breast cancer samples were placed on Visium Gene Expression (GEX) slides. The Visium GEX slides incorporate ~5,000 molecularly barcoded, spatially encoded capture spots onto which tissue sections are placed, stained, and imaged. Following incubation with a human whole transcriptome, probe-based RNA panel and an immuno-oncology oligo-tagged antibody panel, developed with Abcam conjugated antibodies, the tissues are permeabilized and the representative probes are captured. Paired GEX and protein libraries are generated for each section and then sequenced on an Illumina NovaSeq at a depth of ~50,000 reads per spot. Resulting reads from both libraries are aligned and overlaid with H&E-stained tissue images, enabling analysis of both mRNA and protein expression. Additional analyses and data visualizations were performed on the Loupe Browser v4.1 desktop software.ConclusionsSpatial transcriptomics technology complements pathological examination by combining histological assessment with the throughput and deep biological insight of highly-multiplexed protein detection and RNA-seq. Taken together, our work demonstrated that Visium Spatial technology provides a spatially-resolved, multi-analyte view of the tumor microenvironment, where a greater understanding of cellular behavior in and around tumors can help drive discovery of new biomarkers and therapeutic targets.

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Angiopoietin 1 and 2 gene and protein expression is differentially regulated in acute anti-Thy1.1 glomerulonephritis

AJP Renal Physiology ◽

10.1152/ajprenal.00320.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. F1174-F1184 ◽

Cited By ~ 12

Author(s):

Valentina Câmpean ◽

Britta Karpe ◽

Christian Haas ◽

Akram Atalla ◽

Harm Peters ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Glomerular Injury ◽

Double Immunofluorescence ◽

Real Time Quantitative Pcr ◽

Glomerular Capillary ◽

Gene And Protein Expression ◽

Nonradioactive In Situ Hybridization ◽

Angiopoietin 1

Capillary neoformation is important in repair of glomerular injury of various origins. VEGF was shown to be crucial for glomerular capillary repair in glomerulonephritis (GN). We reasoned that other angiogenic factors are likewise involved in glomerular capillary remodeling and found angiopoietin 1 and -2 (ANG1 and ANG2) mRNA to be upregulated in cDNA microarrays of microdissected glomeruli of anti-Thy1.1 GN of the rat. We then studied glomerular in situ gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 in the course of anti-Thy1.1 GN, which was induced by injection of OX-7 antibody. Animals were perfusion fixed at days 6 and 12 after GN induction and compared with nonnephritic controls receiving PBS. Capillary damage and repair were quantitatively analyzed using stereological techniques. Gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 was analyzed using real-time quantitative PCR from microdissected glomeruli, nonradioactive in situ hybridization, double immunofluorescence, and Western blot analysis. Glomerular capillarization assessed as length density was significantly lower at day 6 of anti-Thy1.1 GN than in controls; it was back to normal values at day 12. ANG1 and ANG2 gene expression was markedly upregulated at day 6 of the disease compared with controls. Protein expression of ANG1 and ANG2 was confined to podocytes and that of Tie-2 to endothelial cells. At day 12 of anti-Thy1.1 GN when capillary restoration was nearly completed, ANG1 and ANG2 gene expression returned to basal levels, whereas Tie-2 expression was still high. With the use of a combined molecular and in situ approach, the spatial and temporal gene and protein expression of the angiopoietins and their receptor was analyzed in anti-Thy1.1 GN. The results indicate that glomerular expression of ANG1 and ANG2 and Tie-2 is differentially regulated and may contribute to healing and endothelial cell stabilization in experimental GN.

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Comparison of the Effects of Glibenclamide on Metabolic Parameters, GLUT1 Expression, and Liver Injury in Rats With Severe and Mild Streptozotocin-Induced Diabetes Mellitus

Medicina ◽

10.3390/medicina48100078 ◽

2012 ◽

Vol 48 (10) ◽

pp. 78 ◽

Cited By ~ 5

Author(s):

Jelizaveta Sokolovska ◽

Sergejs Isajevs ◽

Olga Sugoka ◽

Jelena Sharipova ◽

Natalia Paramonova ◽

...

Keyword(s):

Gene Expression ◽

Diabetes Mellitus ◽

Protein Expression ◽

Diabetic Rats ◽

Metabolic Parameters ◽

Gene And Protein Expression ◽

Glut1 Expression ◽

Mild Diabetes ◽

Streptozotocin Induced Diabetes ◽

Glut1 Gene

Background and Objective. Glucose transport via GLUT1 protein could be one of additional mechanisms of the antidiabetic action of sulfonylureas. Here, the GLUT1 gene and the protein expression was studied in rats in the course of severe and mild streptozotocin-induced diabetes mellitus and under glibenclamide treatment. Material and Methods. Severe and mild diabetes mellitus was induced using different streptozotocin doses and standard or high fat chow. Rats were treated with glibenclamide (2 mg/kg daily, per os for 6 weeks). The therapeutic effect of glibenclamide was monitored by measuring several metabolic parameters. The GLUT1 mRNA and the protein expression in the kidneys, heart, and liver was studied by means of real-time R T-PCR and immunohistochemistry. Results. The glibenclamide treatment decreased the blood glucose concentration and increased the insulin level in both models of severe and mild diabetes mellitus. Severe diabetes mellitus provoked an increase in both GLUT1 gene and protein expression in the kidneys and the heart, which was nearly normalized by glibenclamide. In the kidneys of mildly diabetic rats, an increase in the GLUT1 gene expression was neither confirmed on the protein level nor influenced by the glibenclamide treatment. In the liver of severely diabetic rats, the heart and the liver of mildly diabetic rats, the GLUT1 gene and the protein expression was changed independently of each other, which might be explained by abortive transcription, and pre- and posttranslational modifications of gene expression. Conclusions. The GLUT1 expression was found to be affected by the glucose and insulin levels and can be modulated by glibenclamide in severely and mildly diabetic rats. Glibenclamide can prevent the liver damage caused by severe hyperglycemia.

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Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1532-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Neety Sahu ◽

Gaurav Budhiraja ◽

Anuradha Subramanian

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Signaling Pathways ◽

Mesenchymal Stromal Cells ◽

Nuclear Localization ◽

Stromal Cells ◽

Actin Reorganization ◽

Low Intensity ◽

Gene And Protein Expression ◽

Low Intensity Ultrasound

Abstract Background Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

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P4460microRNA-17-5p and microRNA-20a-5p downregulation by atorvastatin in Hepg2 cells: a new mechanism involved in the lipid-lowering therapy

European Heart Journal ◽

10.1093/eurheartj/ehz745.0857 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

K Saavedra ◽

K Leal ◽

D Zapata ◽

S Sagardia ◽

F Ortega ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Hepg2 Cells ◽

Lipid Lowering ◽

Lipid Lowering Therapy ◽

Ldlr Gene ◽

Variable Effect ◽

Incidence And Mortality ◽

Gene And Protein Expression ◽

In Silico Studies

Abstract Background Hypercholesterolemia increases the risk of coronary artery disease and its pharmacological treatment has demonstrated, besides reducing cholesterol levels, a decrease in the incidence and mortality from coronary events. The treatment of hypercholesterolemia is mainly driven by using statins. However, the response to pharmacological therapy shows high inter-individual variability, resulting in a variable effect in both lipid lowering and risk reduction. Thus, a better understanding of lipid-lowering mechanism and response variability at molecular level is required. Previously, we demonstrated a deregulation of microRNA (miR) profile in HepG2 cells after atorvastatin treatment, including the downregulation of miR-17-5p and miR-20a-5p which potentially targets the LDL receptor gene (LDLR), suggesting that it might be involved in allowing the LDLR overexpression on the surface of hepatic cells to subsequently capture circulating LDL and to reach the expected lipid-lowering effect. Purpose To determine the role of miR-17-5p and miR-20a-5p on the regulation of LDLR gene expression in HepG2 cells. Methods Cells HepG2 were treated with atorvastatin 10μM for 24 hours. RNA extraction and enrichment of smallRNAs were performed. The gene expression of miR-17-5p, miR-20a-5p, miR-24-3p, miR-93-5p, miR-106a-5p and LDLR were evaluated. To evaluate the effect of miR-17-5p and miR-20a-5p on LDLR gene expression, both miRs were overexpressed or repressed by transfection of mimics or inhibitors respectively into HepG2 cells for 24, 48 and 72 Hours. The gene expression of LDLR was quantified by real time PCR using RPL27 gene as reference gene. The protein expression of LDLR and beta actin were evaluated using western blot and quantified using the ImageJ software. Results Our data showed that atorvastatin significantly repressed the expression of miR-17-5p (P<0.0001) and miR-20a-5p (P=0.0456) in HepG2 cells. In silico studies showed that miR-17-5p interact with the 3'-UTR region of the LDLR. Consistently, when miR-17-5p or miR-20a-5p were overexpressed by using mimics, we observed that gene and protein expression of LDLR decreased significantly (P<0.0001 and P<0.05 respectively). Consistently, when miR-17-5p or miR-20a-5p were repressed by the use inhibitors, we observed that the gene and protein expression of LDLR increases significantly (P<0.005). Conclusions In conclusion, we demonstrate that atorvastatin induces a significant down-regulation of the miR-17-5p and miR-20a-5p in HepG2 cells. The overexpression or repression regulate the gene and protein expression of LDLR. Acknowledgement/Funding FONDECYT N°3160567, FONDECYT N°1171765 and DIUFRO DI19-0094

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Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression

Diabetologia ◽

10.1007/s00125-020-05290-6 ◽

2020 ◽

Vol 64 (1) ◽

pp. 195-210

Author(s):

Stephany Daniela Villota ◽

Maria Toledo-Rodriguez ◽

Lopa Leach

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Splice Variants ◽

Fold Increase ◽

Lifestyle Changes ◽

Protein Isoforms ◽

Lower Percentage ◽

Systematic Sampling ◽

Regulation Of Expression ◽

Gene And Protein Expression

Abstract Aims/hypothesis Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs). Methods Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 Mr dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured. Results All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 Mr dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold). Conclusions/interpretation Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring.

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Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level

Reproduction ◽

10.1530/rep-14-0019 ◽

2014 ◽

Vol 147 (6) ◽

pp. 789-801 ◽

Cited By ~ 18

Author(s):

M D Saenz-de-Juano ◽

F Marco-Jimenez ◽

B Schmaltz-Panneau ◽

E Jimenez-Trigos ◽

M P Viudes-de-Castro ◽

...

Keyword(s):

Gene Expression ◽

Birth Weight ◽

Protein Expression ◽

Strong Evidence ◽

Foetal Development ◽

Crucial Step ◽

Metabolic Alteration ◽

Gene And Protein Expression ◽

Before And After ◽

First Time

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.

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Mesenchymal Stromal Cells From Myelodysplastic Syndrome Patients Show Lower DICER1 and DROSHA Expression, as Well as Decreased Mir-155 and Mir-181a MicroRNA Expression, When Compared with Healthy Controls

Blood ◽

10.1182/blood.v116.21.397.397 ◽

2010 ◽

Vol 116 (21) ◽

pp. 397-397

Author(s):

Carlos Santamaría ◽

Olga López-VIllar ◽

Sandra Muntión ◽

Belén Blanco ◽

Soraya Carrancio ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Protein Expression ◽

Myelodysplastic Syndrome ◽

Western Blot ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Microrna Expression ◽

Healthy Controls ◽

Lower Expression

Abstract Abstract 397 Mesenchymal stromal cells (MSC) are closely related to the regulation of hematopoietic stem cell niche. Recently, Raaijmakers et al (Nature, 2010), published that deletion of Dicer1, a RNase III enzyme involved in microRNA biogenesis, in murine MSC-derived osteoprogenitors triggered peripherical blood cytopenias, myelodysplasia and subsequent AML, showing that molecular alterations in bone marrow microenvironment could result in clonal impaired haematopoiesis. Here, we have investigated whether MSC from myelodysplastic syndrome (MDS) patients show differences in DICER1 and DROSHA, another RNA III endonuclease, in comparison to healthy MSC. In addition, we have analyzed several hematopoietic-related microRNAs in these same samples. Bone marrow MSC from MDS patients (n=35; 10 5q- syndrome, 4 RA, 5 RARS, 10 RCMD, 3 RAEB, 2 MDS-U and 1 hypocellular MDS) and healthy donors (HD, n=20) were isolated and in vitro expanded following standard procedures until the third passage. Additionally, paired mononuclear cells (MNC) from 13 MDS and 8 HD were obtained. Total RNA was isolated using TRIzol reagent (Invitrogen). DICER1 and DROSHA relative gene expressions were assessed by quantitative PCR (Q-PCR) using commercial TaqMan® assay (Applied Biosystems®) with GAPDH as control gene. DICER1 and DROSHA (Abcam) protein expression were evaluated in whole cell lysates by western blot, using calnexin (Stressgen) as control. Several microRNAs with known role in hematopoiesis and immune system regulation were analyzed in 25 MDS and 12 HD by Q-PCR using commercial TaqMan® MicroRNA assay (Applied Biosystems®) with RNU43 as control microRNA. MSC from MDS showed significant lower DICER1 (0.0035±0.0020 vs. 0.0076±0.0092; p=0.044) and DROSHA (0.0070±0.0028 vs. 0.0135±0.0176; p=0.019) gene expression levels than healthy controls. Moreover, MSC from MDS showed lower protein expression of both DICER1 and DROSHA by western blot analysis, confirming Q-PCR findings. By contrast, no difference in either DICER1 (0.0197±0.0151 vs. 0.0173±0.0112; p=0.9) or DROSHA (0.0089±0.0023 vs. 0.0067±0.0037; p=0.09) gene expression were observed between MNC from MDS and HD. As far as microRNA expression, we observed a lower expression of mir-155 (0.63±0.92 vs. 0.94±0.49; p=0.007) and mir-181a (1.30±0.95 vs. 2.02±1.05; p=0.041) in MSC from MDS in comparison to healthy controls. Mir-155 and mir-181a are involved in T-cell and B-cell differentiation, while mir-155 are also related to erythroid and megakarycytic differentiation. We conclude that MSC from MDS patients show lower expression of DICER and DROSHA, two relevant RNA-III endonucleases involved in the microRNA biogenesis, confirming recent findings in murine models. Moreover, the expression of some microRNA is impaired in these cells, raising the possibility that these microenvironmental alterations could be involved in the MDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

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Baicalein Synergy Lenalidomide To Induce Apoptosis Of Myeloma Cell

Blood ◽

10.1182/blood.v122.21.5328.5328 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5328-5328

Author(s):

Ruibo Zhang ◽

Zi Ma ◽

Shangqin Liu ◽

Li He ◽

Chaoping Xu ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Apoptotic Cell ◽

Time Dependent ◽

Dependent Manner ◽

Rt Pcr ◽

P Values ◽

Combined Application ◽

Gene And Protein Expression ◽

Rpmi 8226

Abstract Objective To understand the apoptotic effects and cereblon (CRBN) gene and protein expression induced by baicalein in MM cells. Methods Apoptotic MM cells induced baicalein，lenalidomide or combination of BAI and lenalidomide were stained by using Annexin-V and analyzed by flow cytometry. RT-PCR was used to detect CRBN gene expression in MM cells. CRBN protein expression was detected by western blot in MM cell lines. Results At the concentration of 40 μmol/L, baicalein can induce apoptosis of U266 cells in a time-dependent manner. At the different BAI treated time points (24h, 48h, 72h), the apoptotic cell percentages were 6.11%, 11.9%, 16.7%; After treated RPMI 8226 cells for 72 hours, combined application of baicalein and lenalidomide (both concentrations are 40 μmol/L) could induce more cell apoptosis than baicalein or lenalidomide alone. The apoptotic cell percentages induced by baicalein, lenalidomide or combined application of baicalein and lenalidomide were 15.9%, 4.27%, and 57.5%. CRBN gene expression detected by RT-PCR could be induced by baicalein in U266 cells in a dose-and time-dependent manner. Treated U266 cells for 24h at concentrations of 10 μmol/L, 20 μmol/L and 40 μmol/L, baicalein upregulated CRBN gene expression times were 2.246 ± 0.068, 2.399 ± 0.178 and 3.591 ± 0.061,respectively，compared to the control group. Statistically, the P values were 0.003, 0.009 and 0.001; Treated U266 cells at concentrations of 40 μmol/L at different time points (6h, 12h and 24h), baicalein upregulated CRBN gene expression times were 2.372 ± 0.079, 2.494 ± 0.189 and 3.228 ± 0.151, its P values were 0.002, 0.008 and 0.002.CRBN protein expression detected by using western blot could be induced by baicalein in both U266 and RPMI8226 cell lines. Conclusions Baicalein at suitable concentrations induced MM cells apoptosis in a time-dependent manner. Comparison with the single component used alone,combined application of baicalein and lenalidomide exhibited stronger inhibition effect on proliferation of RPMI 8226. Considering CRBN is the cellular target for lenalidomide, baicalein can up-regulate the CRBN gene and protein expression in MM cells and may enhance MM cell sensitivity to apoptotic stimuli. Therefore, baicalein up-regulated CRBN gene and protein expression and sensitized MM cells to apoptosis stimuli induced by lenalidomide. It provides us a possibility for baicalein clinical application to overcome the resistance to lenalidomide for MM patients in the future Disclosures: No relevant conflicts of interest to declare.

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