Abstract 13051: Nourin-dependent Mirna-137 : A Novel Early Diagnostic Biomarker for Unstable Angina Patients

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Salwa A Elgebaly ◽  
Robert H Christenson ◽  
Hossam Kandil ◽  
Nashwa El-Khazragy ◽  
Laila Rashed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Unstable Angina ◽  
Cell Damage ◽  
Invasive Coronary Angiography ◽  
Diagnostic Biomarker ◽  
Significant Difference ◽  
Early Biomarker ◽  
Artery Disease ◽  
Spearman’S Correlation ◽  
Early Diagnostic

Introduction: No blood biomarker exits that can diagnose Unstable Angina (UA) patients. Nourin is ischemia-dependent inflammatory mediator rapidly released by reversible ischemic myocardium “before” necrosis, and by necrotic cells. Using Nourin amino acid sequence, Bioinformatics analysis indicated that Nourin is likely regulated by miR-137; a marker of cell damage and a hypoxiaresponsive autophagy-signaling pathway linked to myocardial ischemia and coronary artery disease. Hypothesis: That the Nourin-dependent miR-137, is an early biomarker for UA patients when Troponin levels are below the decision limit. The underlying regulatory mechanism involves lncRCTB89H12.4 and mRNA-FTLH-17; also associated with ischemia. Methods: We measured serum gene expression profile of lnc-CTB89H12.4/miR-137 and mRNAFTHL- 17 in UA (n=30 - confirmed by invasive coronary angiography and negative Troponin) and STEMI (n=16) patients at presentation, and healthy volunteers (n=16). Results: Gene expression of miR-137 was up-regulated by 1,185-fold in UA (median=1,244.41) compared to healthy (1.05), and by 2.5-fold in STEMI (3,162.72) compared to UA (Fig. 1). Receiving Operator Characteristics (ROC) analysis revealed a statistically significant difference in miR-137 that discriminated UA from healthy controls with a test sensitivity & specificity of 96% & 93%, respectively. Diagnostic sensitivity was 75% & specificity was 83% for discriminating UA from STEMI. Additionally, Spearman’s correlation analysis revealed a significant association of miR-137 with lncRCTB89H12.4and mRNA-FTHL-17. The down-regulation of lncR-CTB89H12.4 after ischemia resulted in the up-regulation of miR-137 and activation of mRNA-FTLH-17. Conclusions: As a marker of cell damage, the Nourin-dependent miR-137 is a promising early diagnostic biomarker indicating UA patients and discriminating between UA and STEMI. Regulations appears to be from lncR-CTB89H12.4 and mRNA-FTLH-17.

scholarly journals Nourin-Dependent miR-137 and miR-106b: Novel Early Inflammatory Diagnostic Biomarkers for Unstable Angina Patients

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 368 ◽  
Author(s):  
Salwa A. Elgebaly ◽  
Robert H. Christenson ◽  
Hossam Kandil ◽  
Nashwa El-Khazragy ◽  
Laila Rashed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Unstable Angina ◽  
Healthy Subjects ◽  
Cell Damage ◽  
Expression Profiles ◽  
Invasive Coronary Angiography ◽  
Molecular Networks ◽  
Troponin Level ◽  
Coronary Syndrome ◽  
Mrna Targets

Background: Currently, no blood biomarkers exist that can diagnose unstable angina (UA) patients. Nourin is an early inflammatory mediator rapidly released within 5 min by reversible ischemic myocardium, and if ischemia persists, it is also released by necrosis. Nourin is elevated in acute coronary syndrome (ACS) patients but not in symptomatic noncardiac and healthy subjects. Recently, circulating microRNAs (miRNAs) have been established as markers of disease, including cardiac injury and inflammation. Objectives: To profile and validate the potential diagnostic value of Nourin-dependent miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) as early biomarkers in suspected UA patients and to investigate the association of their target and regulating genes. Methods: Using Nourin amino acid sequence, an integrated bioinformatics analysis was conducted. Analysis indicated that Nourin is a direct target for miR-137 and miR-106b-5p in myocardial ischemic injury. Two linked molecular networks of lncRNA/miRNAs/mRNAs were also retrieved, including CTB89H12.4/miR-137/FTHL-17 and CTB89H12.4/miR-106b-5p/ANAPC11. Gene expression profiling was assessed in serum samples collected at presentation to an emergency department (ED) from: (1) UA patients (n = 30) (confirmed by invasive coronary angiography with stenosis greater than 50% and troponin level below the clinical decision limit); (2) patients with acute ST elevation myocardial infarction (STEMI) (n = 16) (confirmed by persistent ST-segment changes and elevated troponin level); and (3) healthy subjects (n = 16). Results: Gene expression profiles showed that miR-137 and miR-106b-5p were significantly upregulated by 1382-fold and 192-fold in UA compared to healthy, and by 2.5-fold and 4.6-fold in STEMI compared to UA, respectively. Healthy subjects showed minimal expression profile. Receiver operator characteristics (ROC) analysis revealed that the two miRNAs were sensitive and specific biomarkers for assessment of UA and STEMI patients. Additionally, Spearman’s correlation analysis revealed a significant association of miRNAs with the associated mRNA targets and the regulating lncRNA. Conclusions: Nourin-dependent gene expression of miR-137 and miR-106b-5p are novel blood-based biomarkers that can diagnose UA and STEMI patients at presentation and stratify severity of myocardial ischemia, with higher expression in STEMI compared to UA. Early diagnosis of suspected UA patients using the novel Nourin biomarkers is key for initiating guideline-based therapy that improves patients’ health outcomes.

Abstract 407: Use of a Previously Validated Blood-based Test Demonstrates Increased Diagnostic Accuracy as Measured by AUC over Usual Care in the Evaluation of Obstructive Coronary Artery Disease in Males and Females.

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Brian Rhees ◽  
Andrea Johnson ◽  
Alexandra Lansky ◽  
John McPherson ◽  
Matthew Budoff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Coronary Artery Disease ◽  
Diagnostic Accuracy ◽  
Usual Care ◽  
Invasive Coronary Angiography ◽  
Obstructive Coronary Artery Disease ◽  
Blood Gene Expression ◽  
Males And Females ◽  
Artery Disease ◽  
Cad Risk

Background: Current evaluation of stable, non-acute patients presenting with symptoms suggestive of obstructive coronary artery disease (CAD) is costly and often exposes patients to radiation and contrast-dye side effects. These risks are coupled with relative poor diagnostic accuracy, as consistently demonstrated by low yields at invasive coronary angiography. We hypothesized that the use of a previously validated blood-based test incorporating age, sex and whole-blood gene expression, in conjunction with a clinician’s clinical assessment, may improve on usual care methods for the evaluation of these patients. Methods and Results: This analysis includes evaluable data from two prospective multicenter clinical studies [[Unable to Display Character: –]] PREDICT (NCT005617, N=523) and COMPASS (NCT1117506, N=431) where patients were assessed for both pre-test CAD risk according to Diamond-Forrester (D-F) criteria as well as for obstructive CAD using either invasive coronary angiography or cardiac computed tomography angiography (CCTA). All patients in COMPASS were also assessed by myocardial perfusion imaging (MPI); a subset of N=307 subjects were assessed by MPI in PREDICT. Previously, we demonstrated diagnostic superiority, as measured by AUC, for a score combining patient age, sex and whole-blood gene expression (ASGES), in a combined set of men and women from both PREDICT (ASGES = 70%, D-F = 66%, MPI = 54%) and COMPASS (ASGES = 79%, D-F = 69%, MPI = 59%). In this expanded analysis, we report results stratified by sex and demonstrate superiority of the ASGES, as measured by AUC, to MPI for males and females in PREDICT (male ASGES = 66%, MPI = 55%; female ASGES = 65%, MPI = 48%) and in COMPASS (male ASGES = 73%, MPI=60%; female ASGES 73%, MPI = 55% respectively). In addition, we demonstrate that ASGES improves CAD risk classification when compared to D-F criteria in females in both PREDICT (ASGES = 65%, D-F = 51%) and COMPASS (ASGES = 73%, D-F = 58%). Conclusions: We demonstrate that use of a gender-specific, blood-based test incorporating age, sex, and gene expression provides better diagnostic accuracy for patients considered for referral to cardiology and advanced cardiac testing, when compared to usual care methods of D-F type risk classification and MPI.

scholarly journals A Study of Prognostic Value of Hs-CRP and Fibrinogen in Patients of Unstable Angina

2018 ◽  
Vol 7 (1) ◽  
pp. 1609
Author(s):  
Harpreet Singh ◽  
Kunal Bansal ◽  
Tarsem Pal Singh ◽  
Deepa Malik ◽  
Vanita Bansal ◽  
...  
Keyword(s):  
Unstable Angina ◽  
Mean Value ◽  
Clinical Syndrome ◽  
Medical College ◽  
Increased Risk ◽  
Significant Difference ◽  
The Mean ◽  
Hs Crp ◽  
Artery Disease ◽  
Time Of Admission

<p><strong>Background:</strong> Unstable angina constitutes a clinical syndrome that is usually caused by atherosclerotic coronary artery disease and is associated with an increased risk of cardiac death and myocardial infarction.</p><p><strong>Material and Methods:</strong> An open, prospective, observational, comparative study was conducted. The study included 50 cases in age group 20-80 years admitted in Government Medical College, Amritsar diagnosed as unstable angina ruled out by Trop T and CPK-MB at admission. Hs-CRP and Fibrinogen levels were estimated at time of admission and repeated after 48 hours.</p><p><strong>Results:</strong> There was significant difference in the mean value of Hs-CRP between favourable and unfavourable group at the time of admission (0.807±0.37mg/l v/s 3.57±1.70mg/l, p&lt;0.001). A significant difference in the mean value of Hs-CRP was found between favourable and unfavourable group after 48 hours (0.51±0.22mg/l v/s 4.03±1.84mg/l, p&lt;0.001) There was significant difference in the mean value of fibrinogen between favourable and unfavourable group at the time of admission (356.94±72.50mg/dl v/s 588.60±94.89mg/dl, p&lt;0.001). A significant difference in the mean value of fibrinogen was found between favourable and unfavourable group after 48 hours (309.11±75.25mg/dl v/s 622.60±133.42mg/dl, p&lt;0.001).</p><p><strong>Conclusion:</strong> It is concluded that in patients with unstable angina, elevated levels of Hs-CRP and Fibrinogen at admission indicate an adverse hospital outcome.</p>

Abstract 13103: Nourin-dependent Mirna-106b : A Novel Early Inflammatory Diagnostic Biomarker for Cardiac Injury

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Salwa A Elgebaly ◽  
Robert H Christenson ◽  
Hossam Kandil ◽  
Nashwa Elkhazragy ◽  
Laila Rashed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vascular Endothelial Cells ◽  
Inflammatory Marker ◽  
Invasive Coronary Angiography ◽  
Cardiac Injury ◽  
Serum Samples ◽  
Inflammatory Biomarker ◽  
Significant Difference ◽  
Mirna 106B ◽  
Early Diagnose

Introduction: Nourin is an inflammatory mediator rapidly released by ischemic myocardium “before” necrosis, and by necrotic cells . It stimulates leukocyte “ chemotaxis ” and “ activates ” leukocytes and vascular endothelial cells to release several “ cytokine storm ” mediators. Using Nourin amino acid sequence, Bioinformatics analysis indicated that Nourin is likely regulated by miRNA-106b ; an inflammatory-signaling pathway linked to myocardial ischemia. Hypothesis: As an "initial" inflammatory marker, the Nourin-dependent miR-106b can early diagnose ischemia-induced injury in UA patients when Troponin levels are below the decision limit, and in STEMI patients. The underlying regulatory mechanism involves lncR-CTB89H12.4 and mRNA-ANAPC11; associated with ischemia. Methods: Gene expression of lncR-CTB89H12.4 / miR-106b and mRNA-ANAPC11 were measured in serum samples from UA (n=30 - confirmed by invasive coronary angiography and negative Troponin) and STEMI (n=16) patients at presentation, and healthy volunteers (n=16). Results: Gene expression of miR-106b was up-regulated by 150-fold in UA compared to healthy, and by 4.6-fold in STEMI compared to UA (Fig. 1). Receiving Operator Characteristics (ROC) analysis revealed a statistically significant difference in miR-106b that discriminated UA from healthy controls with a test sensitivity of 87% sensitivity & specificity of 88%. Diagnostic sensitivity was 86% and specificity was 90% for discriminating UA from STEMI. Additionally, Spearman’s correlation analysis revealed a significant association of miR-106b with lncR-CTB89H12.4 and mRNA-ANAPC11. The down regulation of lncR-CTB89H12.4 after ischemia resulted in the up-regulation of miR-106b and activation of mRNA-ANAPC11 . Conclusions: The Nourin-dependent miR-106b is a promising early inflammatory biomarker indicating UA patients and discriminating between UA and STEMI. Regulations appears to be from lncR-CTB89H12.4 and mRNA-ANAPC11 .

scholarly journals Diagnosis of coronary heart diseases using gene expression profiling; stable coronary artery disease, cardiac ischemia with and without myocardial necrosis

2015 ◽  
Author(s):  
Nabila Kazmi ◽  
Tom Gaunt
Keyword(s):  
Gene Expression ◽  
Myocardial Infarction ◽  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Unstable Angina ◽  
Stable Coronary Artery Disease ◽  
Myocardial Necrosis ◽  
Cardiac Ischemia ◽  
Myocardial Infraction ◽  
Artery Disease

AbstractCardiovascular disease including coronary artery disease and myocardial infarction is one of the leading causes of death in Europe, and is influenced by both environmental and genetic factors. With the advancements in genomic tools and technologies there is potential to predict and diagnose heart disease using molecular data from analysis of blood cells. We analyzed gene expression data from blood samples taken from normal people (n=21), non-significant coronary artery disease (n=93), patients with unstable angina (n=16), stable coronary artery disease (n=14) and myocardial infarction (MI; n=207). We used a feature selection approach to identify a set of gene expression variables which successfully differentiate different cardiovascular diseases. The initial features were discovered by fitting a linear model for each probe set across all arrays of normal individuals and patients with myocardial infarction. Three different feature optimisation algorithms were devised which identified two most discriminating sets of genes one using MI and normal controls (total genes=8) and another one using MI and unstable angina patients (total genes=17). The results proved the diagnostic robustness of the final feature sets in discriminating not only patients with myocardial infraction from healthy controls but also from patients with clinical symptoms of cardiac ischemia with myocardial necrosis and stable coronary artery disease despite the influence of batch effects and different microarray gene chips and platforms. selection approach to identify a set of gene expression variables which successfully differentiate different cardiovascular diseases. The initial features were discovered by fitting a linear model for each probe set across all arrays of normal individuals and patients with myocardial infarction. Three different feature optimisation algorithms were devised which identified two most discriminating sets of genes one using MI and normal controls (total genes=8) and another one using MI and unstable angina patients (total genes=17). The results proved the diagnostic robustness of the final feature sets in discriminating not only patients with myocardial infraction from healthy controls but also from patients with clinical symptoms of cardiac ischemia with myocardial necrosis and stable coronary artery disease despite the influence of batch effects and different microarray gene chips and platforms.

scholarly journals Oxidative Stress and Homocysteine in Coronary Artery Disease

2001 ◽  
Vol 47 (5) ◽  
pp. 887-892 ◽  
Author(s):  
Viviana Cavalca ◽  
Giuliana Cighetti ◽  
Fabrizia Bamonti ◽  
Alessandro Loaldi ◽  
Luana Bortone ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Coronary Artery Disease ◽  
Lipid Peroxidation ◽  
Coronary Artery ◽  
Unstable Angina ◽  
Stable Angina ◽  
Chronic Stable Angina ◽  
Cardiovascular Patients ◽  
Significant Difference ◽  
Artery Disease

Abstract Background: Oxidative stress is present in cardiovascular diseases (CVDs), and hyperhomocysteinemia, an independent risk factor for these diseases, may play a role by inducing production of oxygen free radicals. Methods: To evaluate the possible role of homocysteine (Hcy) in inducing oxidative stress in coronary artery disease (CAD), plasma Hcy was measured in 68 consecutive cardiovascular patients, and plasma malondialdehyde (MDA), both free and total (free + bound), was measured in 40 patients with CAD (18 with chronic stable angina and 22 with unstable angina). As controls, we tested 70 healthy volunteers. Hcy was measured by an immunoenzymatic method and MDA, an index of lipid peroxidation, by gas chromatography–mass spectrometry. Results: Plasma Hcy concentrations were significantly higher in cardiovascular patients than in controls (10.2 vs 8.9 μmol/L; P &lt;0.0002), with no significant difference between values in the stable and unstable angina subgroups. Similarly, total MDA was significantly higher in the CAD group than in the controls (2.6 vs 1.3 μmol/L; P &lt;0.00001), again with no significant difference between stable and unstable angina patients. By contrast, free MDA, which was significantly higher in the CAD patients than the controls (0.4 vs 0.2 μmol/L; P &lt;0.00001), was also significantly higher in the unstable than in the stable angina group (0.5 vs 0.3 μmol/L; P &lt;0.03). However, no correlation was observed among Hcy and free and total MDA. Conclusions: Our findings show that a moderate increase of Hcy is associated with CVD but that Hcy at the detected values cannot be considered completely responsible for oxidative damage. That lipid peroxidation is involved in CAD is shown by our observation of significantly increased plasma free and total MDA concentrations compared with controls. Moreover, free MDA values discriminated between unstable and chronic stable angina, and could thus represent a new diagnostic tool.

Markers of Hemostatic Activation in Affected Coronary Arteries during Angioplasty

1994 ◽  
Vol 72 (05) ◽  
pp. 672-675 ◽  
Author(s):  
Nicolas W Shammas ◽  
Michael J Cunningham ◽  
Richard M Pomearntz ◽  
Charles W Francis
Keyword(s):  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Venous Blood ◽  
Balloon Inflation ◽  
Blood Samples ◽  
Hemostatic System ◽  
Significant Difference ◽  
Hemostatic Markers ◽  
Artery Disease ◽  
High Doses

SummaryTo characterize the extent of early activation of the hemostatic system following angioplasty, we obtained blood samples from the involved coronary artery of 11 stable angina patients during the procedure and measured sensitive markers of thrombin formation (fibrino-peptide A, prothrombin fragment 1.2, and soluble fibrin) and of platelet activation ((3-thromboglobulin). Levels of hemostatic markers in venous blood obtained from 14 young individuals with low pretest probability for coronary artery disease were not significantly different from levels in venous blood or intracoronary samples obtained prior to angioplasty. Also, there was no translesional (proximal and distal to the lesion) gradient in any of the hemostatic markers before or after angioplasty in samples obtained between 18 and 21 min from the onset of the first balloon inflation. Furthermore, no significant difference was noted between angioplasty and postangioplasty intracoronary concentrations. We conclude that intracoronary hemostatic activation does not occur in the majority of patients during and immediately following coronary angioplasty when high doses of heparin and aspirin are administered.

The Effect of Antiplatelet Drugs on Plasma Betathromboglobulin in Coronary Artery Disease

1979 ◽  
Author(s):  
P Han ◽  
A Turpie ◽  
E Genton ◽  
M Gent
Keyword(s):  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Crossover Trial ◽  
Specific Protein ◽  
Antiplatelet Drugs ◽  
Significant Difference ◽  
Pre Treatment ◽  
Artery Disease ◽  
Therapeutic Doses ◽  
Granule Release

Platelets play a role in the development and complications of coronary artery disease (CAD) and a number of abnormalities of platelet function which can be corrected by antiplatelet drugs have been described. Betathromboglobulin (BTG), a platelet-specific protein which is released from α-granules during platelet activation is significantly elevated in patients with angiographically demonstrated CAD (51.0 ± 31.0 ng/ml., n = 50) compared to normal (28.0 ± 8.0 ng/ml., n = 70) p < 0.001. The effect of sulphinpyrazone (800 mg.) or aspirin (1200 mg.)/dipyridamole (200 mg.) on plasma BTG in CAD was studied in a blind prospective crossover trial in 25 patients. Mean BTG concentration pre-treatment was 52.3 ng/ml. and after 1 month’s treatment with placebo, sulphinpyrazone or aspirin/dipyridamole mean plasma BTG concentrations were 53.5, 49.6 and 56.7 ng/ml. respectively. Analysis of variance showed no significant difference between the means (p > 0.1) . This study confirms increased plasma BTG concentrations in patients with CAD and indicates that therapeutic doses of these antiplatelet drugs do not significantly effect the BTG level and thus appear not to prevent α-granule release in CAD.

Osteoprotegerin and Osteopontin Serum Levels are Associated with Vascular Function and Inflammation in Coronary Artery Disease Patients

2020 ◽  
Vol 18 (5) ◽  
pp. 523-530 ◽  
Author(s):  
Konstantinos Maniatis ◽  
Gerasimos Siasos ◽  
Evangelos Oikonomou ◽  
Manolis Vavuranakis ◽  
Marina Zaromytidou ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Endothelial Function ◽  
Vascular Function ◽  
Serum Levels ◽  
Atherosclerosis Progression ◽  
Control Subjects ◽  
Significant Difference ◽  
Artery Disease ◽  
Stable Cad

Background: Osteoprotegerin and osteopontin have recently emerged as key factors in both vascular remodelling and atherosclerosis progression. Interleukin-6 (IL-6) is an inflammatory cytokine with a key role in atherosclerosis. The relationship of osteoprotegerin, osteopontin, and IL-6 serum levels with endothelial function and arterial stiffness was evaluated in patients with coronary artery disease (CAD). Methods: We enrolled 219 patients with stable CAD and 112 control subjects. Osteoprotegerin, osteopontin and IL-6 serum levels were measured using an ELISA assay. Endothelial function was evaluated by flow-mediated dilation (FMD) in the brachial artery and carotid-femoral pulse wave velocity (PWV) was measured as an index of aortic stiffness. Results: There was no significant difference between control subjects and CAD patients according to age and sex. Compared with control subjects, CAD patients had significantly impaired FMD (p<0.001) and increased PWV (p=0.009). CAD patients also had significantly higher levels of osteoprotegerin (p<0.001), osteopontin (p<0.001) and IL-6 (p=0.03), compared with control subjects. Moreover, IL-6 levels were correlated with osteoprotegerin (r=0.17, p=0.01) and osteopontin (r=0.30, p<0.001) levels. FMD was correlated with osteoprotegerin levels independent of possible confounders [b coefficient= - 0.79, 95% CI (-1.54, -0.05), p=0.04]. Conclusion: CAD patients have increased osteoprotegerin, osteopontin and IL-6 levels. Moreover, there is a consistent association between osteoprotegerin and osteopontin serum levels, vascular function and inflammation in CAD patients. These findings suggest another possible mechanism linking osteoprotegerin and osteopontin serum levels with CAD progression through arterial wall stiffening and inflammation.

ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders

2020 ◽  
Vol 20 (18) ◽  
pp. 2274-2284
Author(s):  
Faroogh Marofi ◽  
Jalal Choupani ◽  
Saeed Solali ◽  
Ghasem Vahedi ◽  
Ali Hassanzadeh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zoledronic Acid ◽  
Mrna Expression ◽  
Promoter Methylation ◽  
Methylation Level ◽  
Target Genes ◽  
Osteoblastic Differentiation ◽  
Therapeutic Effects ◽  
Significant Difference ◽  
Scheduled Time

Objective: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. Materials and Methods: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real.time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. Results: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). Conclusion: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.

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