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3.6.2.1 Circulating miRNAs Expression and Anthropometrics Variables

In this study, none of the six selected circulating miRNAs were associated with anthropometric measurements (weight, BMI, waist, and body fat). It was expected to observe the association of circulating mir-222 and mir-221 with markers of adiposity since mir-222 has been reported to be highly expressed in obese patients while mir-221 was low (Ortega et al., 2013). Surprisingly, the observation of similar expression was not seen in this study. The reason for this could be because the finding of Ortega group was in morbid obesity (mean BMI ≥ 40) while our study population mean BMI was 30.

3.6.2.2 Circulating miRNAs with markers of insulin sensitivity

The key finding of the present study was that among the six circulating miRNAs included, the expression of mir-144 was the only circulating miRNA to display a positive association with HOMA-IR and insulin. Also, among the six selected circulating miRNAs, mir-144 showed the highest association with glucose concentration. All these associations remained significant even after adjusting for age, gender, and body fat.

The level of circulating mir-144 has been found to be increased in patients with T2DM. A meta-analysis of controlled profiling studies showed that the expression of 40 miRNAs were dysregulated in T2DM in several tissues (blood, liver, pancreas, muscle, adipose tissue, and glomeruli) (Zhu & Leung, 2015). One of these 40 miRNAs were circulating mir-144, which was upregulated in human T2DM blood samples. Additionally, Yang et al. showed that the expressions of platelet and plasma mir-144 were altered in T2DM patients with (n = 56) or without (n = 58) ischemic stroke compared to healthy controls (n = 30) (Yang et al., 2015). Moreover, mir-144 appear to contribute to the regulation of insulin signalling. This was supported by the findings of Karolina et al., which showed that elevated circulating mir-144 correlate with the downregulation of insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. Although the participants in this study did not have diabetes, all of them were high-risk patients for developing T2DM, and most of them were IR thus highlighting the potential plasma mir-144 as a key player in insulin resistance which could develop to diabetes over time. This study provides additional evidence for the role of plasma mir-144 in the pathogenesis of T2DM and the possibility of using it as a diagnostic biomarker.

Wang et al. reported a higher expression of circulating miR-144 seems to be ethnicity related; it was only associated with T2DM in native Sweden (Caucasian, n = 68) and not Iraqis patients (Arabs, n = 84) (Wang et al., 2014). In this study, ethnicity was not a factor since all participants were Caucasians.

MiR-221 and miR-222 are two highly homologous miRNAs that have been studied extensively in human malignancies (Song et al., 2017). Few studies have associated the alteration in their expression with diabetes. Ortega et al. reported that circulating mir-222 was highly expressed in Caucasian male patients with T2DM (n = 48) compared to patients with normal glucose tolerance (n = 45) and were associated with fasting glucose and HbA1c (Ortega et al., 2014). Also, insulin administration rapidly altered the expression of circulating mir-222. Shi et al. also reported higher mir-222 expression in the omental adipose tissue of Chines women with gestational diabetes (n = 13) at the time of caesarean delivery compared with pregnant women with normal glucose tolerance (n = 13) (Shi et al., 2014). The authors further used 3T3-L1 adipocytes to demonstrate that stimulation with a relatively high concentration of oestrogen increased mir-222 expression and seems

to negatively regulate adipose insulin sensitivity via suppression of oestrogen receptor α (ER-α) protein and insulin-sensitive membrane transporter glucose transporter 4 (GLUT4) protein. In another study done by Coleman et al. showed that the levels of mir-221 and mir-222, measured in whole tissue homogenates of the internal mammary artery (IMAs) taken from 73 patients undergoing coronary artery bypass surgery, were higher in patients with diabetes compared to normal controls and it remained significantly associated even after adjustment for age, body mass index, and serum lipids (Coleman et al., 2013). Additionally, Meerson and colleagues reported a positive correlation between the expression of mir-221 in subcutaneous adipose sample and BMI, glucose, and insulin in 29 Pima Indian participants without diabetes (Meerson et al., 2013).

Similar association with both mir-222 and mir-221 and glucose, insulin, and HOMA- IR were not observed in this study. Such disparity between the results could be explained by multiple reasons. Firstly, different population studied. The association found between mir-222 and mir-221 and markers of glycemia was in patients with diabetes, whereas the population in this study included patients without diabetes. Secondly, different ethnicity. For example, Meerson et al. study population were Pima Indian participants not suffering from diabetes, one of the highest prevalence of diabetes and obesity of any population in the world (Pearson, 2015), while the participants in this study were Caucasians. A previous study showed that racial/ethnic differences in circulating miRNAs expression between ethnic groups (Wang et al., 2014). Thirdly, different samples (tissue vs. plasma). It is still not known whether miRNAs extracted from cellular or extracellular sample should be utilized interchangeably, and it remains unknown if expression from these distinct sources is suitable. In a large human cohort (2,391 participants) done by Shah et al. showed that whole blood and plasma miRNA expression is distinct and that miRNA expression from different human sources should not be used interchangeably as biomarkers of disease (Shah et al., 2016). For example, in Coleman et al. study, cellular miRNAs (IMAs tissues) were used to measure the expression of miRNAs, which probably demonstrate local intracellular function, while circulating miRNAs (plasma samples) were used in this study which may demonstrate systemic function. Finally, the different methodology used to process samples and measure the expression of miRNAs, which is considered the major factor for the inconsistency between studies.

Meanwhile, the association found in this study between both mir-222 and mir-221 and HbA1c was similar to Ortega et al. findings (Ortega et al., 2014). Glycated haemoglobin (HbA1c) has been considered as a good indicator of overall glycaemic control in 2-3 months. Studies have shown the possibility of using it as a surrogate marker for insulin resistance (Borai et al., 2011; Saha & Schwarz, 2017) which can explain why there was an association between mir-222 and HbA1c and not glucose in the current study.

Adiponectin is an insulin-sensitising and anti-inflammatory adipocytokine, which is considered as a potential mediator between obesity, insulin resistance, and diabetes (Lindberg et al., 2017; Weber et al., 2017). It has been shown that mir- 221 may regulate IR via effects on adiponectin receptor 1 (ADIPOR1). Meerson et

al. displayed that mir-221 directly downregulates ADIPOR1 expression suggesting

that it may be possible that obesity-associated upregulation of mir-221 can result in a decrease in ADIPOR1, which may, in turn, lead to the development of obesity- related metabolic consequences such as insulin resistance or T2DM (Meerson et

al., 2013). In another study done by Hwang et al., showed that there was a

negative correlation between mir-221 expression and ADIPOR1 (Adiponectin receptor 1) mRNA expression in breast tumours as well as in breast cancer cell lines (Hwang et al., 2013). In the present study, total plasma adiponectin was negatively associated with the expression of circulating mir-221. In multiple linear regression models adjusting for age, gender, and body fat didn’t change the association. The result demonstrates that the association between circulating adiponectin and the expression of circulating mir-221 is independent of obesity.

Upregulation of the expression of circulating mir-192 and mir-193b in the pre- diabetes state was previously reported by Párrizas et al. (Párrizas et al., 2015). Their study included 92 participants (29 healthy control, 22 pre-diabetes with impaired fasting glucose (IFG), 21 prediabetes with impaired glucose tolerance (IGT), and 20 newly diagnosed T2DM). Out of the 176 circulating miRNAs they tested (most frequently detected in human serum/plasma), circulating levels of three miRNAs (mir-150, mir-192, and mir-193b) achieved high levels in both the IFG and IGT groups but were not altered in patients with T2DM. Interestingly, they also found that mir-192 and mir-193b decrease to normal in insulin resistant humans after therapeutic lifestyle intervention consisting of a monitored exercise

program and diet recommendations. Also, Jaeger et al. did a longitudinal study where they assessed the association of four circulating miRNAs, including mir-192 with incident T2DM (Jaeger et al., 2018). The study included 213 patients from the Vorarlberg Institute for Vascular Investigation and Treatment (VIVIT) cohort who didn’t have T2DM at baseline and followed for six years. Patients who developed diabetes (n = 35) had higher levels of mir-192 compared to those who did not (n = 178). In agreement with previous findings, the results of this study showed that both mir-192 and mir-193b were positively correlated with glucose in patients without diabetes but high-risk, and most of them were IR. In contrast to the findings of the present study, Ortega et al. reported decreased levels of mir- 192 in the serum of patients with T2DM (Ortega et al., 2014).

Furthermore, the expression of mir-155 was positively associated with glucose, HbA1c. In a study done by Lin et al., showed that downregulation of mir-155 levels was found in serum from patients with T2DM (n = 30) compared to healthy controls and showed a negative correlation with HOMA-IR suggesting that mir-155 might be involved in glucose homeostasis and insulin action. (Lin et al., 2016). Moreover, mir-155 expression in PBMCs (peripheral blood mononuclear cells) from patients with T2DM (n = 20) was decreased compared to normal controls (n =20) (Corral- Fernández et al., 2013). A possible reason for these variations could be the different population studied and different samples used.

3.6.2.3 Circulating miRNAs and liver enzymes

In this study, the expression of both mir-192 and mir-193b were associated with liver enzymes (ALT and GGT) independent of age, sex, and body fat. Párrizas et

al. also reported a good correlation between mir-192, mir-193b and FLI (Fatty

Liver Index, a mathematical index calculated from blood TAG, BMI, waist circumference, and hepatic enzyme GGT and has been used as a substitution for hepatic steatosis). Jaeger et al. reported a positive correlation between serum levels of mir-192 and AST, ALT, and GGT using baseline samples.

3.6.2.4 Circulating miRNAs and inflammatory biomarkers

Increased levels of mir-155 have been associated with various inflammatory diseases, including rheumatoid arthritis (RA) (Alivernini et al., 2018), and it is

considered as a potential regulator of inflammation. It plays a key role in the immune response, and studies have demonstrated its involvement in the development of atherosclerosis (Li et al., 2016; Faccini et al., 2017). C-reactive protein (CRP) is a sensitive, nonspecific marker for systemic inflammation and a powerful predictor for cardiovascular diseases (Koenig, 2013). A recent study included 132 patients who underwent coronary angiography to measure coronary flow, 66 patients with coronary slow flow and 66 patients with normal coronary flow showed that plasma mir-155 was positively correlated with CRP (Su, Yang & Li, 2018). In the present study, there was no association between mir-155 and both CRP and WBC although. A possible reason for these variations could be the different population studied and different samples used.