Population
The resent case-control study was conducted on 134 Iranian subject who performed coronary angiography at Hazrat-e-Rasoul Hospital, Iran University of Medical Sciences. 65 patients with CAD and 67 healthy controls recruited in this study between April 2016 and February 2017. The diagnosis of CAD was according to coronary angiography. A cardiologist evaluated all the angiograms and the presence of more than 50% stenosis in at least one major coronary artery categorized as CAD [15]. In addition, participants with less than 25% stenosis in all the coronary artery were considered as healthy controls.
Participants with history of cancer, diabetes, renal failure, liver diseases, and any other chronic diseases and those taking immunosuppressive, steroids medications, and anti-inflammatory during last six months were excluded from the study. Also, subjects who had unstable angina, carotid plaque and any history of cardiovascular disease, including cerebrovascular, acute coronary syndrome, and peripheral artery disease were excluded from control group. Medical history, demographic data and medications consumption of the studied subjects were collected through a questionnaire. Basic anthropometric data including systolic and diastolic blood pressure, weight, and height were measured. Ethics approval was obtained from the Research Ethics Committee of Iran University of Medical Sciences and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki (IR. IUMS.REC1395.9221184201).
Biochemical parameter measurements
Fifteen mL of venous blood were collected after an overnight fasting (8 to 12 hours) and stored at -80°C. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and fasting blood glucose (FBG) levels were measured, using commercially available kits (Pars Azmon kit, Iran).
PBMCs isolation and RNA-Extraction
Ten mL EDTA-containing whole blood tubes was used for PBMCs isolation, by Ficoll-Hypaque density-gradient centrifugation as previously described [16]. Total RNA was extracted from the cell lysates, using the miRNeas mini kit (QIAGEN, USA) according to manufacturer’s instructions. The purity and concentration of extracted RNA were evaluated via 1.5% agarose gel electrophoresis (Biorad, US) and the nanodrop spectrophotometer (thermo-scientific, US).
cDNA synthesis and Quantitation of mRNA
Gene expressions analysis was performed using real time PCR. In the first step, 1 µg total RNA were converted to cDNA, by Revert Aid First cDNA Synthesis Kit (Takara, Japan). Real time PCR was run in an ABI-Step One (Applied Biosystems, USA) system, using RealQ Plus 2x Master Mix Green (Amplicon, Denmark). GAPDH was used as internal control. Specific primers used to amplify genes are listed as follows: GAPDH: F: 5΄ CCC CTT CAT TGA CCT CAA CTA C 3ʹ, R: 5 ʹGAT GAC AAG TTC CCG TCT C 3΄; MMP − 9, F: 5΄ CCT GGG CAG ATT CCA CT, R: 5 ΄CCA AGT GTT CCG AGT AGT TTT GGA 3; TIMP1, F: 5΄ACT GCA GGA TGG ACT CTT GCA3, R: 5TTT CGA AGC CTT GGA GGA GCT 3΄.
Statistical Analysis
SPSS version 20 was used for the statistical analysis of the data. Continuous variables were shown as mean ± standard deviation and tested by Student t-test or Mann–Whitney U according to normality test. Categorical data tested using chi-square test and represented using frequency and percentage. Spearmen’s correlation test was applied for the correlation between continuous variables. ROC was applied to test the diagnostic ability of the desirable variables. A p value less that 0.05 considered as statistically significant.