Relationship of hypomethylation CpG islands in interleukin-6 gene promoter with IL-6 mRNA levels in patients with coronary atherosclerosis

Introduction: Atherosclerosis is the important cause of most cardiovascular diseases, with high prevalence and mortality. Atherosclerosis is not only a lipid metabolism disorder but also recently is defined as a chronic inflammatory disease. Several studies showed that interleukin-6 (IL-6) is involved in the pathogenesis of atherosclerosis. The aim of the present study is the examination of IL6 mRNA Levels and hypomethylation of IL6 promoter in atherosclerosis patients. Methods: In this assay, a total of 35 cases with atherosclerosis and 30 controls were enrolled. RNA and DNA were isolated from the peripheral blood of all samples. Mean IL6 gene expression was determined by RT-PCR and methylation status at six CpG motifs in IL6 promoter was determined using bisulfite genomic sequencing. Results: Real Time-PCR analysis results showed the mean IL6 RNA level in atherosclerosis patients candidate for CABG (coronary artery bypass grafting) was significantly higher than controls (P value = 0.01). Also, the upstream CpG motifs (-1038 to -952) in IL6 promoter were predominantly unmethylated in patients than in the controls (P value = 0.01). Conclusion: These findings suggest that an increase in IL-6 gene expression and its DNA hypomethylation promoter are associated with atherosclerosis patient’s candidate for CABG surgery.


Introduction
Atherosclerosis is the leading cause of cardiovascular disease, including ischemic gangrene, coronary heart diseases, heart failure, abdominal aortic aneurysm, and stroke. 1 It is responsible for the highest mortality in Western societies, which is increasing significantly in developing countries. 2 Recent data suggest that the most common risk factor for the adult Iranian population is atherosclerosis. [3][4][5] The most important health problem in Iran is the high prevalence and mortality rate. 6,7 Atherosclerosis develops over a person's lifetime and involves several steps: Endothelium activation and immune cells employment; the differentiation of monocytes and the formation of foam cell, fibrotic plaques formation due to foam cells death and proliferation and migration of SMCs (smooth muscle cells); and plaque rupture and thrombosis. 2,8 It has now been well accepted that atherosclerosis is not only a lipid disorder but also chronic inflammatory disease. 9,10 Inflammation is known as an important factor in the development of atherogenesis through adverse effects on the metabolism of lipoprotein and arterial wall biology. Both innate and acquired immune system has been implicated in the atherogenic process. [9][10][11] In atherosclerosis, both immune responses (the innate and adaptive) are organized by a wide range of cytokines that control all of the disease stages. Up to now, a various group of cytokines, low-molecular-weight proteins has been identified. Cytokines are divided into several classes, such as the interleukins (IL), tumor necrosis factors (TNF), chemokines, transforming growth factors (TGF), the interferons (IFN), and colony-stimulating factors (CSF). 8,9,12 A large body of evidence suggests that interleukin signaling promotes atherogenic activity. 13 Interleukin-6 is an uncommon cytokine with antiinflammatory and pro-inflammatory characteristics, a culprit in the development and complications of atherosclerotic disease. 14 IL-6 has deep effects on the spread and activation of T cells. The cell differentiation of CD8 + T cells into cytotoxic T Cells is promoted with IL6 15 In lymphoid tissue, IL-6 alone or in association with other cytokines stimulates differentiation of T follicular helper (Tfh) cells, T helper 1 and 17. [15][16][17] IL-6, together with growth factor (TGF) -β, plays an important role in the differentiation of Th17 cells from naive CD4 + T cells. 15,18,19 Naive T CD4+ cell precursors are generated Tfh and they participate in antigen-specific B lymphocytes differentiation into memory and plasma cells. 15,20 Among atherosclerosis patients, the response of systemic inflammatory variability can be explained by different patterns of epigenetic changes that alter the expression of genes involved in the process of the atherosclerotic process.
Mechanisms that modify gene expression without changing the underlying gene sequence are collectively referred to as epigenetic, although that this term is typically used for changes that are heritable through mitosis. Epigenetic changes are sometimes transmitted from parent to child, although it is controversial what role they play in transgenic epigenetic changes in human life. At the DNA level, the underlying mechanisms of short-and long-term regulation of gene expression are overlapped. Epigenetic mechanisms, such as histone modification, DNA methylation, and non-coding RNA can regulate gene expression while the underlying DNA sequence remains the same. 21 DNA methylation is a significant epigenetic mechanism of the regulation of transcription. 22 Methylation modification is an alternative mechanism regulating IL-6 production 23,24 and the consistent evidence has revealed that IL-6 expression is associated with a reduction in the DNA methylation of its gene promoter. 25 The aim of the current study was firstly, to assay the IL-6 mRNA Levels in the atherosclerosis patients' peripheral blood, secondly, to examine the methylation status of IL-6 promoter in atherosclerosis patients who are candidate for coronary artery bypass grafting surgery, and finally to evaluate the relationship between methylation status of CpG motifs in IL-6 gene promoter and IL-6 mRNA levels in these patients.

Study sample
A total of 35 patients with atherosclerosis were recruited from Department of Cardiac Surgery, Afshar Hospital, Yazd, Iran. The sample size was determined at the significance level of α = 0.05 using PASS (Power and Sample Size) software (version 5) and the procedure of comparing the means of two independent groups. The diagnosis of atherosclerosis was established by a cardiologist during the angiographic evidence. The coronary arteries wrap around the entire heart. The left coronary artery (LM) has two main branches called the anterior descending (LAD) and the circumflex (LCX) or rotating artery, and there is a right coronary artery that is called RCA. In a case, if the right coronary artery or one of the branches of the left coronary artery has a blockage of more than 50%, the case is 1VD. If there are two coronary arteries with a blockage of more than 50%, the case is 2VD and the patients who have three coronary arteries with a blockage of more than 50% are 3VD. There was no coronary artery with significant blockage (less than 50%) in control case. As a control group, 30 cardiovascular disease-free subjects, determined by physical examination, history analysis, electrocardiography, and echocardiography were recruited from the same hospital during the same period when the case-patients were recruited. The controls were frequency-matched to the cases by age (±5 years) and sex. For both atherosclerosis and control groups, subjects with hypertension, diabetes, peripheral artery disease, autoimmune-related disease or cancers were excluded. Information on age, sex, height, weight, cigarette smoking, and family history of cardiovascular disease was obtained using a structured questionnaire through in-person interviews. The clinical data and characteristics for the current study are summarized in Table 1. Patients and control cases were divided into four groups: CADgroup with normal coronary artery, and CAD + group with significant lesions (>50% narrowing of luminal diameter) in one, two, or three vessels (LAD, LCX, and RCA). All of the patients were candidates for CABG (Coronary Artery Bypass Grafting) surgery.  26 Touchdown PCR was performed in a 20 μL reaction volume containing 7 μL converted DNA by sodium bisulfite, 10 μL of PCR Master mix (2×), 2 μL of forward and reverse primers (2.5 mM), and 1 μL sterile distilled water. The Touchdown PCR was done under the following condition: initial denaturation stage at 95°C for 5 min, followed by 6 cycles of 95°C for 1 minute, 55-58°C for 50 seconds, and 72°C for 50 seconds, followed by 35 cycles of 95°C for 1 minute, 53°C for 50 seconds, 72°C for 50 seconds, and final extension at 72°C for 5 minutes. The forward and reverse primers for this PCR reaction were 5'-GGTTTTTGAATTAGTTTGATT-3' and 5-CCCTATAAATCTTGATTTAAAAT-3' respectively. 27 For the detection of PCR products, 2% agarose gel and staining with ethidium bromide were used. Methylation level was expressed as methylated cytosine percent on total methylated and non-methylene cytosines. Non-CpG cytosine residues are used as built-in controls to check bisulfite conversion. The values were expressed as the mean for all the sites and separately for six CpGs at the IL-6 gene promoter.

Statistical analysis
Variables were tested in triplicate, and experiments were repeated at least twice. Z-score standardized values were used for both mean of CpG island methylation values and 2 -∆Ct , excluding outliers from analyses. Pearson correlation and t Student test were carried out using (SPSS) software (IBM SPSS 22, SPSS Inc., Chicago, IL., USA) and values of P < 0.05 were considered statistically significant. 28

Results
The chi-square test was performed for each trait between the two patients and control groups. At first, the percentages for all the studied traits were calculated in two groups of patients and normal cases, and it was evaluated the qualitative variables in both groups. The chi-square and the P values were displayed in each comparison in Table 1. P values for all of them were more than 0.05. They indicate that there is no significant difference between the two groups in terms of gender, age, sex, smoking, and family history of cardiovascular disease. Therefore, the selection of samples for this study was done correctly and the involvement of other factors such as these has been minimized in the results of the study.
The mRNA expression levels of different genes in patients with atherosclerosis and healthy subjects were determined by RT-qPCR technique. The quantitative data of IL6 and HPRT1 genes expression by the RT-PCR were also analyzed by SPSS software. To do this, after calculating the mean and standard deviation, t test was performed on the data ( Table 2). The t-test is used to compare the mean of two independent groups and it proves statistically the existence or absence of a significant difference between the two groups. In the first part of the Table 2, the results of the Levene's test can be seen. This test shows whether the variance of the IL-6 gene expression for the two groups (patient and control) is the same or not. The results of the Levene's test determine which SPSS outputs will be correct for use. If the value of Sig. in Levene's test be greater than 0.05, the data in the first row of the table must be used, because they indicate that the variances of the two study groups are the same (Equal variance assumed). While if the value of Sig. be less than 0.05, it means that the variances are not the same and the data in the second row of the table (Equal variance not assumed) must be used.
As it is seen in Table 2, the value of Sig. in the Levene's test is equal to 0.167. This indicates the same variances in IL-6 gene expression in both the patient and control groups. In other words, the two groups are homogeneous, and each on follows a normal distribution curve. Therefore, the conclusion of this data can be reliable.
The qPCR analysis showed that the IL-6 mRNA expression level in patients with atherosclerosis was significantly higher than that in controls (P < 0.05). Among those who had coronary plaques, patients with 3VD, 2VD, and 1VD showed more levels of the IL-6 mRNA expression in whole blood than controls, respectively. Figure 1 illustrates that the average of IL-6 gene expression in 3VD patients is the highest, and it is the lowest in controls. IL-6 gene expression level was also associated with its DNA methylation pattern. The region of the IL-6 gene promoter from nucleotide -1083 to -952 contains 6 CpG motifs ( Figure 2). The methylation status of this region was compared in patients with atherosclerosis and control groups. According to bisulfite sequencing results ( Figure  3), the six CpG motifs in this region were predominantly methylated or semi-methylated in controls, while CpG motifs at -1041C, -1038C, and -1036C in patient group were predominantly unmethylated (P < 0.05), and the CpG motifs at -1013C, -1008C, and -1004C were methylated  Two arbitrary groups were defined according to the mean levels of IL-6 mRNA expression, and the methylation of the 6 CpG motifs between -1083 and -952 was compared in both healthy controls and atherosclerotic patients. The results were similar to those obtained previously (Figure 4). The CpG motifs at -1041C, -1038C, and -1036C were predominantly methylated in cases showed lower IL-6 mRNA expression levels (controls and some of 1VD patients), while these motifs were unmethylated or semimethylated in cases with higher levels of mRNA expression (2VD and 3VD patients), and it showed a significant difference (P < 0.05). 29 The status of methylation in CpG motifs at -1013C, -1008C, and -1004C was similar and both arbitrary groups were methylated (P > 0.05) ( Figure 5).
The correlation between methylation and gene expression level in the two groups of patients and control was measured by Pearson correlation coefficient test. The results of this test are shown in Table 3. According to it, the correlations between positions -1041C, -1038C, and -1036C and gene IL6 mRNA levels are negative in patients and positive in control cases. The p-values prove the correlations are significant in both groups (correlation is significant at Sig. less than 0.05). Therefore, by increasing the expression of IL6 gene, all these three positions have shown a decrease in methylation status in patients, and by decreasing the IL6 mRNA levels, all these three positions have indicated an increase in methylation status in controls.
The correlations between methylation status of positions -1013C, -1008C, and -1004C and gene expression in the two studied group were not significant (correlation is not significant at Sig. more than 0.05).
The results of sequence alignment of IL-6 gene among numerous species presented that these six CpG motifs were located in highly conserved regions among several species (human, gibbon, cow, chimpanzee, elephant, mouse, dog, rat, chicken, and zebrafish).

Discussion
Interleukin-6, a pleiotropic cytokine, regulates a variety of inflammatory responses. The formation of atherosclerotic plaques is caused by the accumulation of many genetic and epigenetic molecular changes. Previous studies were shown that IL-6 level is raised in CAD patients and may be an inflammation marker for cardiovascular risk. 30 The results of Lefkou et al displayed that children (5-15 years) with a family history of premature coronary artery disease meaningfully increased IL-6 plasma levels compared to the control group of pediatric cases without a family history (P < 0.001). 31 The findings of Fisman et al showed that people with stable coronary artery disease had higher IL-6 with a weaker prognosis over an average follow-up of about 6 years. In their study, the increase of IL-6 (each 1 pg/mL) was associated with increased relative odds 1.70 (range 1.23-2.45) of following sudden death. 32 In research in China, with 263 patients with ST-segment elevation myocardial infarction, the admission level of IL-6 was correlated with cardiovascular mortality for more than 3 years of follow-up. 33 Additionally, Murine experiments, with a number suggest that they do not protect the infarction damage with the elimination of IL-6 effects. 34 However, there is little information about the profile of CpG spots and their methylation status in the promoter of the IL-6 gene in patients with atherosclerosis. In our study, a total of six CpG motifs in the IL-6 promoter was determined using bisulfite genomic sequencing in the

Iranian population.
An active process is DNA methylation that catalyzed by DNA methyltransferases in cytosine residues in CpG dinucleotides. The previous studies suggested that the expression of these enzymes was influenced by several factors including cigarette age, sex, and smoking. These studies show that the mRNA level of DNA methyltransferase was higher in smokers than in nonsmokers. Also, in human cells and tissues, an age-dependent decrease of the methylation levels of DNA cytosine-5-methyltransferase and methylation was shown. 35 The findings of Zuo suggested that DNA hypomethylation of IL-6 promoter is associated with the increased risk for acute myocardial infarction.
According to the results of the transcription regulation database to identify transcription factors DNA binding to these six CpG motifs, the importance of these CpG motifs are highlighted for controlling transcription rate and gene expression ( Table 4). The methylated or unmethylated status will affect the binding of transcription factors to the DNA sequence. The status of DNA methylation is lower in transcriptionally active genes, and our results are consistent with the concept that methylation is important in controlling the expression of IL-6 as seen in atherosclerosis. All of the participants in this study were exclusive of the average age of the elderly, so the generalization of our findings to young people or other races/ethnic groups is uncertain. We measured only interleukin-6 with expression levels from blood taken during a physical examination, but the IL-6 concentration may change over time. It should be pointed out that our findings do not prove a direct causal relationship between IL-6 promoter hypomethylation and atherogenesis, but just describe an association between these two processes.

Conclusion
Our study reported a significant increase in IL-6 gene expression in whole blood in patients with atherosclerosis than controls. These findings suggest that DNA hypomethylation of IL-6 gene promoter and increased mean IL-6 gene expression are associated with the risk of atherosclerosis. Other mechanisms might be involved in the regulation of gene expression that needs further investigations. To confirm the causal relationship between TF-DNA binding and methylation, additional laboratory tests such as analyzing motif mutations in specific loci and assessing their impact on the local DNA methylation changes are needed.