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Chapter 21 Micro RNAs for Prediction of Clinical Outcomes in CAD JAYAGOPAL P.B. • S.K.M. SARJUN BASHA

Drosha processing) has also been reported, and these are known as mirtrons. Both pre-miRNAs and mirtrons are actively transported to the cyto-plasm by the Guanosine Triphosphate (GTP)-dependent transporter, Exportin 5. Within the cytoplasm, pre-miRNAs are further cleaved by Dicer (another RNase III enzyme), generating un-stable double-stranded miRNA duplexes which contain a functional miRNA ‘guide’ strand and a ‘passenger’ strand. Subsequently, the duplex is un-wound and the passenger strand degraded, leaving the guide strand to enter the RNA-induced silenc-ing complex (RISC). Mature miRNA associates it-self to the Argonaute proteins and thus enters the RISC. miRNA binds to complementary sequences in the 3’-untranslated region (UTR) of target mRNA within the RISC which then targets the RISC to the specifi c mRNA to induce post transcriptional gene silencing through translational inhibition and/or mRNA degradation leading to miRNA-mediated downregulation of the corresponding target pro-tein 1 . A single miRNA can regulate the expression of several genes depending on the specifi city of the target sequence. Conversely, individual genes can be regulated by different miRNAs if they carry complementary sequences for more than one miRNA – these factors lead to a highly complex regulatory mechanism 2 , 3 .

miRNA AND ATHEROSCLEROSIS

Endothelial cell (EC) dysfunction is a key step in the initiation of atherosclerosis. Schober et al. 4 demonstrated that one of the critical steps in main-taining a proliferative reserve of ECs in response to shear stress is mediated by miR-126-5p is through suppression of delta – like homologue 1 (Dlk1). Thus, the diminished levels of miR-126-5p can re-duce the proliferative reserve of ECs, thereby pro-moting plaque formation. Also, miRNA-155 causes

Coronary artery disease (CAD) is both a leading cause of death and an important health threat worldwide including Asian countries. Progression of CAD is highly variable and is infl uenced by both environmental factors and genetic determi-nants. The genetic risk occurs due to unfavourable combinations of genetic variations in multiple genes that have not yet been completely character-ized. Various blood markers for CAD have been identifi ed, but only few have diagnostic or clinical implications.

Thus, innovative early and reliable biomarkers that can assess the risk for CAD and early processes of atherosclerosis are desirable. Considering this, it has been postulated that circulating micro ribonu-cleic acids (miRNAs) could be a useful biomarker of various diseases including CAD.

miRNA

miRNAs are a class of 20–25 nucleotide single-stranded noncoding RNAs that negatively regulate cellular function through modulating messenger RNA (mRNA) degradation and repress translation of mRNAs that contain complementary sequences. miRNA regulate the gene expression regarding cell growth, proliferation and apoptosis. miRNA are seen in a stable protected form in the body tissues, plasma and other body fl uids. Thus, the presence of miRNA in tissue and plasma may act as a poten-tial biomarker in diagnosis, for therapeutic effi -ciency and in prognosis of specifi c conditions. miRNA genes are transcribed by RNA polymerase II into molecules approximately 2 kb long called pri-mary miRNAs. Within the nucleus, these are cleaved into precursor miRNAs (pre-miRNA) by Drosha, an RNase III enzyme, in association with DGCR8, an RNA-binding protein. Interestingly, the presence of pre-miRNAs that are processed by direct splicing of introns (and thereby bypassing

164 SECTION III — Coronary Artery Disease

an increased permeability of ECs leading to infi ltra-tion of infl ammatory cells. miR-143/145 complex has been shown as a critical regulator of vascular smooth muscle cell differentiation.

Xiao et al. 5 analysed vascular endothelial enriched miRNA (miR-126) in plasma from 31 patients with CAD and 36 patients without CAD by quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). They concluded that miR-126 was signifi -cantly decreased in patients with CAD and high low-density lipoprotein (LDL) cholesterol and miR-126 was signifi cantly increased when LDL cholesterol was high in patient who had risk factors for CAD but did not have angiographically signifi cant CAD. This reduction of endothelially expressed miRNAs may be attributed to uptake into atherosclerotic lesions within vasculature of CAD patients. Thus, plasma levels of endothelium-enriched circulating miR-126 could be a potentially useful as a sensitive biomarker in assessing endothelial damage as it is substantially altered among patients with suspected CAD (or) sub-jects with coronary risk factors.

SMOKING, ENDOTHELIAL FUNCTION AND miRNA

The role of miRNA as biomarkers of endothelial damage was also studied in middle-aged male smok-ers 6 . In smokers, endothelial function expressed as reactive hyperaemia peripheral arterial tonometry (RH-PAT) index and high-density lipoprotein (HDL) cholesterol levels was inversely correlated with se-rum cotinine level, a metabolic marker of nicotine. On the other hand, it was positively correlated with non-HDL cholesterol and insulin resistance at 8 weeks. In smokers who completely attained smok-ing cessation, both RH-PAT index and plasma miR-126 values were increased when compared with subjects who could not attain smoking cessation. These results suggest an association between vascu-lar endothelial damage in middle-aged male smok-ers and dysregulated plasma miR-126 levels. An increase in miR-126 levels were correlated with fa-vourable effect of smoking cessation and recovery of endothelial function.

PLATELETS AND miRNA

Edelstein et al. 7 studied the racial differences in hu-man platelet PAR4 reactivity. They reported that PAR4 thrombin receptor-mediated platelet aggregation was greater in black subjects. The platelets in these sub-jects showed lower levels of miR-376c, which contrib-uted to higher impression of phosphatidylcholine

transfer protein involved in platelet activation through thrombin which induces dysfunction of ECs through NF-�B and contributes to formation of arterial throm-bus. Thus, it implies that the genomic contribution to platelet function may differ by race and this explains lower survival of black individuals as compared to white individuals after myocardial infarction due to coronary atherothrombosis.

miRNA IN CAD

Endothelial expressed miRNA – miR-126, miR-92a, miR-17, miR-145, miR-155, miR-208 and miR-133a – are decreased in CAD patients 8 .

Smooth muscle-enriched miR-145 is reduced in CAD patients.

Cardiac muscle-enriched miRNAs – miR- 33, miR-208a – are increased in CAD patients 9 .

CAD patients have increased levels of miR-221 and miR-222 in endothelial progenitor cells (EPCs) and these miRNAs bring about mobilization of EPCs 9 , 10 .

Oxidative stress defences in human EPCs are regulated by miR-21, thereby indirectly regulating the condition of CAD. Antagonism of miR-21 im-proves dysfunctional angiogenic progenitor cells in CAD patients.

miRNA IN ACUTE CORONARY SYNDROME

In the largest study by Devaux et al. 11 , the levels of six miRNAs in 1155 patients attending the emer-gency departments with acute chest pain were mea-sured; 244 patients were diagnosed with acute myo-cardial infarction (AMI). However, while the level of miR-208b, miR-499 and miR-320a were signifi cantly higher in patients with AMI compared with other diagnosis, they were not comparable to cardiac tro-ponin T (cTnT) or high-sensitive cTnT (hsTnT) in diagnostic ability. They also reported that while miR-208b moderately predicted survival at 30 days, none of the miRNAs were able to predict long-term mortality (2 years).

CONCLUSION

miRNA act as a biomarker useful for diagnosis and evaluation of pathophysiology of CAD. Shortcom-ings are as follows:

(1) Understanding miRNA-based regulation of gene expression. Single miRNA has multiple targets; hence, it is necessary to fi nd out the exact

165Chapter 21 — Micro RNAs for Prediction of Clinical Outcomes in CAD

mechanism and pathway. Therefore, the role of individual miRNAs and its important targets needs to be defi ned in different settings based on cell types and pathological conditions.

(2) Standardization in samples, sample processing, RNA extraction and storage needs care in the laboratory.

(3) Extensive research and multicentric investiga-tion based on much larger and multi-ethnic patient populations are required to explore the diagnostic values of miRNA as successful as con-ventional biomarker for CAD.

REFERENCES

1. Bartel, D. P. ( 2004 ). MicroRNAs: Genomics, biogenesis, mechanism, and function . Cell , 116 , 281 – 297 .

2. Condorelli, G., Latronico, M. V., & Cavarretta, E. ( 2014 ). MicroRNAs in cardiovascular diseases: Current knowledge and the road ahead . Journal of American Col-lege of Cardiology , 63 , 2177 – 2187 .

3. Kumarswamy, R., & Thum, T. ( 2013 ). Non-coding RNAs in cardiac remodeling and heart failure . Circula-tion Research , 113 , 676 – 689 .

4. Schober, A., Nazari-Jahantigh, M., Wei, Y., Bidzhekov, K., Gremse, F., Grommes, J. , et al . ( 2014 ). MicroRNA-126-5p promote endothelial proliferation and limits atherosclerosis by suppressing Dlk1 . Nature Medicine , 20 , 368 – 376 .

5. Sun X, Zhang M, Sanagawa A, Mori C, Ito S, Iwaki S, et al. (2012). Circulating microRNA-126 in patients with coro-nary artery disease: correlation with LDL cholesterol. Thrombosis Journal , 10, 16.

6. Sugiura, T., Dohi, Y., Yamashita, S., Iwaki, S., Ito, S., Sanagawa, A. , et al . ( 2015 ). Circulating level of microRNA-126 may be a potential biomarker for recov-ery from smoking-related vascular damage in middle-aged habitual smokers . International Journal of Cardiology , 7 , 83 – 87 .

7. Edelstein, L. C., Simon, L. M., Montoya, R. T., Holin-stat, M., Chen, E. S., Bergeron, A. , et al . ( 2013 ). Racial differences in human platelet PAR4 reactivity refl ect expression of PCTP and miR-376c . Nature Medicine , 19 , 1609 – 1616 .

8. Contu, R., Latronico, M. V., & Condorelli, G. ( 2010 ). Circulating microRNAs as potential biomarkers of cor-onary artery disease: A promise to be fulfi lled? Circula-tion Research , 107 ( 5 ), 573 – 574 .

9. Fichtlscherer, S., De Rosa, S., Fox, H., Schwietz, T., Fischer, A., Liebetrau, C. , et al . ( 2010 ). Circulating mi-croRNAs in patients with coronary artery disease . Cir-culation Research , 107 ( 5 ), 677 – 684 .

10. Jamaluddin, M. S., Weakley, S. M., Zhang, L., Kougias, P., Lin, P. H., Yao, Q. , et al . ( 2011 ). miRNAs: Roles and clinical applications in vascular disease . Expert Review of Molecular Diagnostics , 11 ( 1 ), 79 – 89 .

11. Devaux, Y., Mueller, M., Haaf, P., Goretti, E., Twerenbold, R., Zangrando, J. , et al . ( 2015 ). Diagnostic and prognostic value of circulating microRNAs in patients with acute chest pain . Journal of Internal Medicine , 277 , 260 – 271 .