The current study recruited 1064 participants, including 533 ischemic stroke (IS) patients and 531 unrelated healthy controls. 533 IS patients were recruited from the Department of Neurology, First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Guangxi, China. 531 healthy controls were recruited from the physical examination center in the same hospital. All the research participants were Chinese Han people and not related.
The inclusion and exclusion criteria of IS patients and controls were as follows: IS patients were diagnosed by at least 2 experienced doctors based on CT or MRI results, and excluded due to other serious illness, such as hemorrhagic stroke, cancer, and severe shock. Controls were healthy individuals without stroke, coronary heart disease and other cardiovascular diseases. Before being included in this study, each participant provided informed consent. This study was approved by the medical ethics committee of the Guangxi University of Chinese Medicine.
Collection of related clinical parameters
The 2ml plasma was collected through anticoagulant blood collection tube. The StAgO Capact coagulation meter was used to detect the parameters of coagulation function in plasma, including activated partial prothrombin time (APPT), D dimer (D-D), fibrinogen (FIB), international normalized ratio (INR), prothrombin time (PT), prothrombin time activity (PTA), Thrombin Time (TT).
DNA Isolation and Genotyping
The blood genomic DNA was extracted from the peripheral blood of each participant by using an blood genomic DNA extraction kit (Aidlab biotechnologies CO. Ltd) according to the instructions. Subsequently, the DNA samples were qualified and stored in -80 ℃ until use. Genotyping was performed by Bomiao Biological Co., Ltd. (Beijing, China) through Sequenom MassARRAY technology platform (Sequenom, San Diego, CA, USA). Specific primers for genotyping were designed by AssayDesigner 3.1 software and as follows: F: 5’-ACGTTGGATGCAAGCCCATTCCACAGACTC-3’ and R: 5’-ACGTTGGATGATTCTCCACATGGTGTGCTG-3’. For quality control, 5% DNA samples were randomly selected for double testing without the knowledge of the inspectors. The repeatability of these DNA samples reaches 100%, which confirms the reliability of genotyping.
Detection of SRC expression
The SRC expression was detected in the peripheral blood from some research objects, including 50 IS patients and 50 healthy controls. Total RNA was extracted from peripheral blood using the Trizol reagent (Invitrogen,Carlsbad, CA, USA). cDNA was synthesized through the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara Bio Inc.) according the instructions. Quantitative real-time polymerase chain reaction (qPCR) was conducted to detect the SRC expression using the SYBR® Premix Ex TaqTM II kit (Takara Bio Inc.). The related primer sequences were as follows, GAPDH (housekeeper gene) primer: F: 5’-GGTGGTCTCCTCTGACTTCAACA-3’ and R: 5’-GTTGCTGTAGCCAAATTCGTTGT-3’; SRC primer: F: 5’-AGACTGGGCTCTGGCTCTGTTC-3’ and R: 5’-TTGGCAAGATGCCACAAACTG-3’ . The relative expression of SRC was calculated using 2−ΔCT method.
The human embryonic kidney 293T cells (HEK-293T cells) was purchased from Chinese Academy of Sciences, Shanghai. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995065) containing 10% fetal bovine serum (FBS) (Gibco, 10099141C) and 1% streptomycin and penicillin (Gibco, 15140122) at 37 ℃ under 5% CO2 condition.
Construction and transfection of luciferase reporter plasmid
SNPs located in the 5’UTR region were reported to have the potential to regulate parental gene expression . Whether rs6017916 regulate the SRC expression? To clarify this question, we constructed rs6017916 wild type (WT: A) and mutant type (MT: C) into the upstream 5’UTR region of the firefly luciferase coding region. The constructs were further confirmed by sequencing (Shanghai Genechem Co., Ltd). The constructed firefly luciferase and Renilla luciferase vectors were transfected into HEK-293T cells through the X-tremegene HP reagent kit (Roche, 6366244001). Cells were collected 48h after transfection for luciferase activity detection.
Luciferase Activity Assays
Firefly luciferase and Renilla luciferase activity were tested with Dual-Luciferase® Reporter Assay System (Promega, E2910) according to instructions. Renilla luciferase activity was used to normalize firefly luciferase activity to obtain relative luciferase activity.
PLINK software (http://pngu.mgh.harvard.edu/~purcell/plink/) was used for genetic association analysis. The Chi-square goodnessof-fit test was used to evaluate Hardy-Weinberg equilibrium. The comparison of genotype distribution and allele frequency between groups was performed using the Chi-square test. Unconditional logistic regression model analysis was used to evaluate the association between rs6017916 and IS susceptibility under multiple genetic models (additive, dominant, and recessive models). The association between rs6017916 and related clinical parameters was evaluated using general linear model. Moreover, the comparison of SRC expression and related luciferase activity between groups was statistically analyzed by SPSS software (17.0). Statistical significance was set at P < 0.05, and all tests were two-tailed.