The latest research show that miRNA can play an important role as a biomarker for diabetes and its complications [3]. The potential for biofluids–derived miRNA to serve as a diagnostic tool has stimulated a wide range of study regarding the disease-specific expression of miRNA and its stability [33]. In order to find the unique miRNA profile in T2DM patients with IHD, we used a state-of-the-art NanoString nCounter platform that provides the opportunity to profile a large number of miRNAs that have not been previously investigated in relation to IHD in diabetes. In comparison to other methods of miRNA detection, the NanoString offers greater sensitivity and specificity with high quality of data due to the elimination of amplification [34]. It has been shown that this platform detects miRNAs in biofluids with sensitivity and specificity greater than other miRNA detection methods like Real-Time PCR or microarrays. [35]. This makes NanoString as a best strategy to identify novel biomarker candidates for IHD prognosis. To our best knowledge, this study is the first miRNA profiling in the serum of IHD patients using NanoString platform.
Our research revealed 9 miRNAs differentially expressed in patients with T2DM and IHD in comparison to diabetic individuals without IHD. Among the differently expressed miRNAs, several were previously described as associated with the IHD or T2DM. For instance, it has been shown that platelet miR-615-5p was upregulated in patients with IHD as compared to healthy patients. The activation of platelets and the formation of a thrombus on a ruptured atherosclerotic plaque is an important mechanism in the pathogenesis of IHD [36]. Another research showed that the level of miR-1303 in serum was significantly increased in T2DM patients. Importantly, the level of miR-1303 was higher in serum of patient with microvascular complications (neuropathy, nephropathy, and retinopathy) than those without complications [37]. Fittingly, our results indicated upregulation of miR-615-3p and miR-1303 in the serum of T2DM IHD (+) patients. No significant expression of miR-615-3p and miR-1303 was observed in T2DM without IHD. These results suggest that miR-615-3p and miR-1303 are common for T2DM IHD (+) phenotype and could play a pivotal role in the IHD diagnosis in diabetic patients. Chen et al. has shown that miR-548d-3p regulates the expression of the ERBB2 gene. This gene encodes an oncogenic HER2 tyrosine kinase receptor, highly expressed in various types of human tumours [38]. Moreover, ERBB2 has an essential role in cardiac function and development [39, 40]. Clinical studies have indicated that downregulation of ERBB2 leads to heart dysfunctions [41]. Correspondingly, our results showed significant overexpression of miR-548d-3p in T2DM IHD (+) patients. Given the involvement of miR-548d-3p in regulation of ERBB2, required for the proper functioning of the heart, it is plausible to expect miR-548d-3p to be a potential biomarker for IHD. We expect that the rest identified miRNAs candidates (miR-498, miR-1224-5p, miR-3147, miR-548b-3p, miR-651-5p and miR-4455) may also have a high probability of being specific for diabetic patients with IHD.
The area under the ROC curve remains a major criterion for diagnostic biomarkers [32]. To the best of our knowledge, there is no evidence in the literature describing the usefulness of miR-1224-5p, miR-1303, miR-3147, miR-498, miR-548d-3p, miR-615-3p, miR-548b-3p, miR-651-5p and miR-4455 as diagnostic tool for IHD in T2DM. Interestingly, miR-1303 in plasma was tested as a biomarker for acute myocardial infarction [42]. The AUC for miR-1303 was 0.884, but it was lower than AUC for high-sensitive cardiac troponin I (hs-cTnI) which seems to be a more specific biomarker in MI. In this study, the highest AUC values were observed for miR-1224-5p, miR-1303, miR-3147 and miR-498. Furthermore, other tested miRNA (miR-548d-3p, miR-615-3p, miR-548b-3p, miR-651-5p) also showed high AUC results. High AUC scores for these miRNAs provide the groundwork for future confirmatory studies with a comprehensive validation in a larger cohort of patients. Additionally, AUC values for MIF and CXCL12, which play an important role in IHD, were below 0.5, what disqualifies these two proteins as a good diagnostic tool for IHD (+) diabetic patients. MIF as a proinflammatory factor is involved in processes and disorders such as atherosclerosis and myocardial infarction [43, 13]. It is also considered as a potential biomarker for heart diseases in patients with T2DM [44]. Similarly, CXCL12 has an important role in cardiovascular dysfunctions. Research shows that levels of this chemokine are prognostic tools to the prediction of future cardiovascular risk in patients with myocardial infarction, IHD or heart failure [45–47]. However, one has to consider that diabetes is also an inflammation state and non-specific inflammatory parameters like MIF or CXCL12 can be elevated in patients either with IHD or without it. Low AUC values for those proteins support our observations that indeed the changes in miRNA levels could be better prognostic IHD biomarkers than the level of MIF or CXCL12 in 2TDM patients.
We could not find any statistically significant correlation between the level of miRNAs and clinical parameters such as BMI, platelet levels, fasting glucose, HbA1c or triglycerides. However, those parameters are not specific only for ischemic heart disease. Interestingly, we indicated a positive moderate correlation between fibrinogen and miR-1303, miR-3147, miR-498, miR-548d-3p. Fibrinogen is not only an indicator of hypercoagulability but, as an acute phase protein, is also an indicator of inflammation. In epidemiological and clinical studies, elevated blood fibrinogen levels have been shown to be an independent risk factor for cardiovascular diseases [48–50]. It has been proven that miRNAs are able to regulate fibrinogen production [51]. In our study, we indicated that the relationship between levels of fibrinogen and four miRNAs (miR-1303, miR-3147, miR-498, miR-548d-3p) that have not been previously described as being related to this protein. A strong or moderate positive correlation between all miRNAs level suggested they may belong to the miRNA group which participates in the dysregulation of mechanisms responsible for diabetic complications.
IPA analysis indicated 858 molecules regulated by identified miRNAs. Further analysis of these genes showed their connection with cardiovascular diseases, including IHD. Among genes regulated by miRNA were apolipoptotein B and MEF2A. The apolipoprotein B encoded by APOB, potentially regulated by miR-615-3p, is showing association with risk of ischemic heart disease [52]. The same miRNA regulates the MEF2A gene. MEF2A encodes transcription factor, that has an important role in the differentiation of cardiomyocytes. Also, it has been demonstrated that MEF2A is involved in the homeostasis of cardiac cells [53]. Studies show that NAMPT, possibly regulated by miR-548b-3p, is one of the major genes involved in hypertrophy of the heart [54]. Hypertrophy is known as a disease that can lead to IHD and other cardiovascular dysfunctions [55]. On the other hand, TBX5 (T-box transcription factor) has also a crucial role in the development of cardiac disorders. TBX5 is potentially regulated by miR-4455 and it is considered as a key regulator of heart development [56]. Similarly, it was indicated that Scm Polycomb Group Protein Like 4, encoded by SCML4, participates in atherosclerosis mechanism that can lead to ischemic heart disease [57]. In our study, IPA analysis has shown, that miR-1224-5p is involved in the regulation of SCML4 gene. This analysis pointed out considerable relationship between tested miRNAs and genes involved in cardiovascular dysfunctions.
Ingenuity core analysis allowed us to identify which canonical pathways are dysregulated by tested upregulated miRNAs. Our results showed that one of the most important dysregulated canonical pathways in the IHD (+) T2DM patients was apelin cardiomyocyte signalling pathway. Apelin is an endogenous peptide widely expressed in cardiomyocytes, brain, pancreatic islets and adipose tissue [58]. Under normal conditions, it is involved in lowering of blood pressure [59]. Recent studies have found that this apelin-mediated signalling is connected to heart failure and ischemic heart disease. Furthermore, apelin participates in the pathology of diabetes by playing a key role in increasing glucose uptake and insulin sensitivity [60]. Another important dysregulated signalling pathway was atherosclerosis signalling. Atherosclerosis is the process, when plaque builds up inside arteries underlying pathology of cardiovascular diseases and can lead to myocardial infarction [61]. Next altered pathway presented in our study was connected with granzyme B. Granzyme B is an enzyme of the serine protease family and is able to induce cell apoptosis by activating intracellular caspases. Apoptosis induced by granzyme B plays an essential role in cardiovascular diseases such as atherosclerosis, IHD and myocardial infarction [62]. Moreover, it was shown that in patients with chronic kidney disease, granzyme B can be a predictive factor of ischemic heart disease [63]. Additionally, plasma levels of granzyme B are increased in patients with acute coronary syndrome [64]. Endothelin-1 signalling was another canonical pathway highlighted by IPA analysis. Endothelin-1 is a potent vasoconstrictor and pro-inflammatory protein and is an important contributor to the pathogenesis of hypertension, atherosclerosis, hypertrophy and diabetes [65]. In normal conditions, endothelin-1 is involved in increase arterial blood pressure and reduction of heart rate. Influenced by risk factors for cardiovascular disorders its expression is altered, what plays an important role in the pathology of the cardiovascular system [66]. Furthermore, patients with T2DM have increased vasoconstrictor activity induced by endothelin-1 [67]. Certainly, these identified canonical pathways play a significant role in the mechanisms leading to the development of cardiovascular diseases. It allows to suppose that determined by us miRNAs play a relevant role in the regulation of genes associated with cardiovascular diseases and top dysregulated canonical pathways.