Patients and control subjects
This case-control study was carried out on 200 RRMS patients referred to Fars Multiple Sclerosis Society and 200 healthy controls. Patients who had chronic inflammatory disorders, cancer, autoimmune disease, drug intake etc. were excluded. All the patients were in the relapsing state. Furthermore, healthy controls had no autoimmune disease in themselves as well as their family members. Individuals in the case and control groups were age- and sex- matched. Patients were diagnosed as having MS based on the modified McDonald criteria [19, 20] and the disability score of patients was determined by Expanded Disability Status Scale (EDSS) . In this study, MS subjects were selected from those in the remitting state that had received no immunomodulating medications for at least 3 months before sampling. The protocol of this study was approved by the Human Research Ethics Committee from the Shiraz University of Medical Sciences, Fars, Iran and written informed consent forms was taken by all subjects. Blood samples from MS patients were collected when the disease was clinically diagnosed. To perform experiments, 10 ml of venus blood from all participants was collected in EDTA tubes via venipuncture.
Real-time PCR genotyping of SNPs
Using Real-time allelic discrimination Taq-Man assays (Applied Biosystems, Foster City, USA), patients and controls were genotyped for rs9904341, rs17878467, and rs8073069 SNPs in BIRC5 gene. The reactions mixture contained 5 μl of genomic DNA (200 ng/μl), 5 μl of Taq-Man Master Mix (Applied Biosystems, Foster City, USA), 0.5 μl of Taq-Man Genotyping Assay mix containing FAM or VIC labeled probes and primers (Applied Biosystems, Foster City, USA), and H2O to reach a final volume of 25 μl. Real-Time allelic discrimination PCR condition that was performed via StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, USA) was: initially 60 °C for 30 s and then 95 °C for 10 min, and then 40 amplification cycles in 95 °C for 15 s and 60 °C for 1 min, and ultimately 60 °C for 30 s.
PBMC separation, RNA extraction, cDNA synthesis
To attain PBMCs from 50 RRMS patients and 50 healthy individuals, the Ficoll-Hypaque density gradient approach was done. To isolate the RNA, the MiRNeasy Mini kit (Qiagen, Germany) was used. For determination of the yield and purity of isolated RNAs, a Nano Drop spectrophotometer at 260/280 nm (Nano Drop ND-2000C Spectrophotometer, Thermo Fisher Scientific, USA) was used. The first strand complementary DNA (cDNA) was synthesized using the miScript II RT Kit (Qiagen, Germany) based on the manufacturer’s protocol.
Real-time PCR quantification of miRNA and survivin expression
Measurement of the miRNA expression levels (including miR-16, miR-34a, miR-150, and miR-203a) was carried out by miScript SYBR Green PCR Kit (Qiagen, Germany) and StepOne Plus Real-Time PCR (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained 6 μl cDNA template, 10 μl of SYBR Green PCR Master mix, 2 μl primers each, and RNase free water to a total volume of 25 μl. The Real-time quantitative PCR conditions were: initial 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 55 °C for 30 s, and finally 65 °C for 30 s.
Real-time PCR quantification of survivin mRNA level in PBMCs from RRMS patients and healthy controls was performed using RealQ Plus Master Mix Green High ROX (AMPLIQON, Denmark) and StepOne Plus Real-time PCR machine (Applied Biosystems, Foster City, CA, USA). The components of the mixture were 10 μl SYBR Green Master Mix, 8 μl cDNA, 0.4 μl primers each (forward primer of 5′-CCACCGCATCTCTACATTCA-3′ and reverse primer of 5′-GTCTGGCTCGTTCTCAGTGG-3′; adopted from Primer Bank; https://pga.mgh.harvard.edu/primerbank/), and RNase free H2O for a final volume of 25 μl. The thermocyclic conditions of Real-time PCR were: the holding step of 95 °C for 15 min, 50 cycles of 95 °C for 15 s, and 63 °C for 30 s, and then 70 °C for 1 min.
Comparative CT method was exploited to calculate relative miRNA and survivin expressions as previously described by Livak and Schmittgen . For normalizing the expression levels of target genes, the transcript levels of RUN6 (for miRNAs) and GAPDH (for survivin), as the housekeeping genes, were determined.
To determine the survivin level, the serum samples were isolated from the peripheral blood of 50 patients and 50 control subjects. Survivin level was determined using enzyme-linked immunosorbent assay (ELISA) and a commercial kit (Human Survivin ELISA Kit, OriGene Technologies, Inc., Rockville, MD, USA).
Genotype and allelic distribution between case and control groups were implemented by Chi-Square test. Pearson’s χ2-tests were applied to test for significance differences of both genotype and allele frequencies between two groups. The odds ratio (OR) and 95% confidence interval (CI) were calculated. The genotype distributions of chosen SNPs were tested for deviation from Hardy-Weinberg equilibrium in case and control. Determination of normality of scale data distribution was conducted using the Kolmogorov-Smirnov test. The independent t-test or ANOVA was used to compare the groups. The GraphPad Prism v. 6.00 (GraphPad Software, Inc., San Diego, CA, USA, www.graphpad.com) was exploited for plotting. SPSS software v. 21 (SPSS, Chicago, IL, USA) was used for data analysis. Data were presented as number and percentage or mean ± standard deviation (SD) with statistical significance at P < 0.05.