1 Study of circulating microRNA -499 As A Potential Molecular Marker For Acute Myocardial Infarction in Humans Amal Abou El -Fadle MD, Osama S. Arafa… [602348]

1 Study of circulating microRNA -499 As A Potential Molecular
Marker For Acute Myocardial Infarction in Humans

Amal Abou El -Fadle MD, Osama S. Arafa MD *, Adel F. Al -Kholy
MD, Naglaa F. Al Husseini MD and Shaymaa M. Abd El Rahman
M.Sc

Departments of Medical Biochemistry & Cardiology, Faculty of
Medicine, Benha University, Egypt

Abstract
Objectives: To assess gene expression of circulating miRNA -499
using real time PCR technique as early molecular marker for
myocardial injury .
Patients & Methods: The study in cluded 15 patients with acute
myocardial infarction (AMI group) and 20 patients with chronic stable
coronary artery disease (St. CAD) and 15 volunteers (Control group).
All patients underwent coronary catheterization and angiography.
Severity of coronary s tenoses was assessed using Gensini score.
Venous blood samples were obtained for routine laboratory
investigations, ELISA estimation of creatine kinase -MB (CK -MB)
isozymes concentration and serum positivity for cardiac troponin I
(cTnI) and for real time P CR detection and quantification of miRNA –
499.
Results: All AMI and 3 St. CAD patients were positive for cTnI.
Serum CK -MB levels and miRNA -499 relative levels in AMI patients
were significantly higher than controls and St. CAD patients. Relative
miRNA -499 quantity level was increased among AMI patients by
3.187 and 2.202 fold versus controls and St. CAD patients. Relative
miRNA499 levels showed positive significant correlation with
Gensini score, serum positivity for cTnI, CK -MB and total cholesterol
levels. Statistical analyses defined relative miRNA499 level at 11.2
and 12.1 RU as sensitive and specific predictors of high Gensini score,
respectively.
Conclusion: Circulating levels of miRNA -499 could differentiate
between AMI and St. CAD patients. Estima ted miRNA499 at range of
11.2-12.1 RU showed high diagnostic value and specific for diagnosis
of AMI, so can predict the severity of cardiac insult as early non –
invasive diagnostic modality.

2 Introduction
Myocardial infarction (MI) is a major cause of mor tality and
morbidity worldwide (Jneid et al., 2013) . The universal definition
differentiates patients with MI due to plaque rupture from those due to
myocardial oxygen supply -demand imbalance secondary to other
acute illnesses (Chapman et al., 2016) .
Diagno stic criteria of acute myocardial infarction (AMI)
requires clinical symptoms, detection of altered cardiac biomarkers '
levels , new ischemic electrocardiographic changes, or typical imaging
findings indicating myocardial necrosis (Eggers et al., 2009) . Car diac
troponins (cTn) I and T are the cornerstone of diagnostics of AMI .
However, elevat ed plasma cTn levels showed certain limitations
(Jarolim, 2014) with rhythmic diurnal variation of cTnT that is not
seen with cTnI (Klinkenberg et al., 2016) .
MicroRNAs (miRNA) are small, non -coding, single -strand
RNA molecules (Xu et al., 2012) that are found in the great majority
of eukaryotic cells (Papoutsidakis et al., 2014) . MiRNAs negatively
regulate gene expression via translational inhibition or mRNA
degradation followed by protein synthesis repression (Xu et al.,
2012) , thus miRNAs have emerged as key regulators of many
physiological and pathological processes (Bertero et al., 2013) .
In humans, more than 2000 mature miRNAs have been
identified and about one-third of human protein -encoding genes seem
to be miRNA regulated (Kato & Natarajan, 2015) . Expression of
miRNAs, in humans, appears to exhibit a tissue – or cell -specific
distribution (Mayr et al., 2013) . Cardiac and skeletal muscle -specific
miRNA could be relea sed from injured muscle to circulation (Pleister
et al., 2013) , so circulating miRNAs could act as novel biomarkers for
diverse cardiovascular diseases, including AMI (Xu et al., 2012) .
Early decision -making for patients with suspected AMI can
improve out come and decrease mortality, so processes to shorten time
for definitive diagnoses are of value (Peacock et al., 2016) . MiRNA –
208 family (mRNA -499, miRNA -208a, and miRNA -208b ) are among
cardiac specific miRNAs (Li et al., 2013) . The current study aimed to
assess the gene expression of circulating miRNA -499 usi ng real time
PCR technique as early molecular marker for myocardial injury.

Patients & Methods
This prospective comparative study was conducted at
Cardiology and Medical Biochemistry Departments; in conjunction
with Molecular Biology and Biotechnology Unit. Study protocol was

3 approved by the Local Ethical Committee. All studied patients signed
written fully informed consent by themselves or by near relatives prior
to inclusion in the study. The study included 15 patients with acute
myocardial infarction (AMI group) diagnosed according to Thygesen
et al., (2007) and 20 patients with chronic stable coronary artery
disease (St. CAD) and had no recent acute attack, but had previous
angiographic examination (St. CAD group). Fifteen healthy volunteers
with no previous history of chest pain and normal ECG were enrolled
as Control group.
Patients with history of previous cardiac surgery, muscle
compartmental syndrome, recent surgical procedures or trauma
espec ially if involving muscle tissue, received intravenous
thrombolytic or anticoagulant therapy before the initial blood samples
donation, were excluded from the study.
Patients' demographic data including age, gender, weight,
height for calculation of body mass index (BMI = weight (kg)/height
(m2) were determined. Patients were assessed for coronary risk factors
as obesity, smoking, hypertension, hypercholesterolemia and diabetes
mellitus. All patients underwent coronary catheterization and
angiography. CAD was defined as presence of one or more coronary
stenoses with >50% lumen narrowing. Then, the severity of coronary
artery stenosis was assessed using Gensini score (Gensini, 1983) .

Blood sample collection:
Peripheral venous blood samples were obtained from control
subjects, St CAD patients and AMI patients prior to any manipulation.
Blood samples were divided into 3 parts:
1. The first part was sent for hospital emergency lab for complete
blood counting, estimation of serum creatinine, aspartate
aminotrans ferase (AST), total cholesterol, high -density lipoprotein
cholesterol (HDL -c) and triglycerides (Tg).
2. The second part was collected in a plain tube, allowed to clot and
centrifuged at 3000 rpm and sera were stored at -80oC till ELISA
estimation of cTnI lev el (Heeschen et al., 1999) and creatine
kinase -MB (CK -MB) isozymes (Landesberg et al., 2003) .
3. The third part was collected in vacutainer tubes containing EDTA
and stored at -80oC till molecular assay of microRNA according to
manufacturer's instructions (Wang et al., 2012) . Gene fold
expression changes are calculated using the equation 2-∆∆CT
(Livak & Schmittgen, 2010) .

4 Statistical analysis
Results were analyzed using One -way ANOVA with post -hoc
Tukey HSD Test and Chi -square test (X2 test). Sensitivity &
specificity of estimated parameters as predictors were evaluated using
the receiver operating characteristic (ROC) curve analysis judged by
the area under the curve (AUC) compared versus the null hypothesis
that AUC=0.05. Regression analysis (Stepwise method ) was used for
stratification of studied parameters. Statistical analysis was conducted
using the IBM SPSS (Version 23, 2015) statistical package. P value
<0.05 was considered statistically significant.

Results
The study included 15 AMI patients and 20 p atients had St.
CAD. For AMI patients, time lapsed between onset of oppressing
chest pain sensation and blood sampling at emergency department was
81.3±31; range: 40 -130 minutes. There was non -significant (p>0.05)
difference between patients of studied gro up and control group as
regards enrolment data. However, the frequency of associated medical
diseases per affected patient was significantly (P=0.038) higher
among AMI patients than controls. Mean Gensini angiographic score
was significantly (P=0.001) high er in AMI patients than St. CAD
patients. Estimated serum levels of AST, total cholesterol, LDL,
triglycerides were significantly higher with significantly lower serum
HDL in patients compared to controls and in AMI patients than in St.
CAD patients (Table 1).
Eighteen patients were positive for cTnI; 15 AMI and 3 St.
CAD patients with significantly (<0.001) higher frequency among
AMI patients. Serum CK -MB levels (170.9±105 IU/L) in AMI
patients were significantly (=0.001) higher compared to controls
(12.1±2.9 IU/L) and St. CAD patients (20±3.5 IU/L) with non –
significantly (p>0.05) higher levels in St. CAD patients than controls.
Mean circulating miRNA -499 relative level in AMI patients
(12.57±0.4 relative unit [RU]) was significantly (P=0.001) higher tha n
levels estimated in control (11.21±0.28 RU) and St. CAD (11.57±0.35
RU) groups (Fig. 1). The relative miRNA -499 quantity (RQ) level was
increased among AMI patients by 3.187 and 2.202 fold than controls
and St. CAD patients, while was increased by 1.447 fold in St. CAD
patients than controls (Fig. 2).
Development of AMI among the studied cardiac patients
showed positive significant correlation with patients' age (r=0.425,
p=0.001), Gensini angiographic score (r=0.900, p<0.001), serum

5 levels of AST (r=0 .488, p=0.003), total cholesterol (r=0.531,
p=0.001), Tg (r=0.494, p=0.003) and CK -MB (r=0.746, p<0.001) ,
serum positivity for cTnI (r=0.712, p<0.001) , relative miRNA499
circulating level (r=0.808, p<0.001), while showed negative
significant correlation wi th serum level of HDL -c (r= -0.327, p=0.027).
Estimated relative levels of miRNA499 showed positive significant
correlation with Gensini angiographic score (r=0.724, p<0.001),
serum positivity for cTnI (r=0.635, p<0.001), serum CK -MB level
(r=0.541, p=0.001 ) and serum total cholesterol (r=0.347, p=0.041).
Regression analysis defined high Gensini angiography scoring,
high circulating miRNA499 relative level, low serum HDL -c and high
serum total cholesterol as the significant predictors for presence or
liabili ty to develop AMI, in decreasing order of significance.
Evaluation of various cutoff points for circulating miRNA499
relative level as non -invasive predictor for severity of cardiac lesion as
judged by Gensini angiography scoring showed that circulating
miRNA499 relative level at range of 11.2 -12.1 RU could define high
Gensini score indicating positive angiographic pathology with
specificity and PPV of 100%, sensitivity in rang of 75 -80% and
disease prevalence rate ranging between 42.86 and 57.14%. ROC
curve analysis defined circulating miRNA499 relative level at cutoff
points of 11.2 and 12.1 RU as sensitive (Fig. 3) and specific (Fig. 4)
predictor of high Gensini angiographic score, respectively (Table 2).

Table (1): Enrolment data of studied patients a nd controls

Group
Data Control
(n=15) AMI
(n=15) St. CAD
(n=20)
Age (years) 54±10.6 51.3±9.7 56±7.9
Gender Males: Females 10:5 13:2 15:5
Positive family history 1 (6.7%) 4 (26.7%) 4 (20%)
Smoking status Non: Ex: Current smoker 6:5:4 3:4:8 5:6:9
Weight (Kg) 88.4±7.8 91.5±8.8 91.9±12.1
Height (cm) 171.9±4.5 170.5±3.9 169.5±3.5
BMI (Kg/m2) 49.9±2.6 31.5±3.4 31.9±3.5
Other
morbidities No: Yes 7:8 3:12 8:12
Frequency/affected patient 1.5 2.17* 1.92
Gensini angiographic score 0 49±8.3† 23±4.5
Laboratory
investigations Serum AST (mg/dl) 30.1±6.6 55.6±14.8*† 40.8±12.5*
Hb. concentration (gm%) 12.1±1.3 12.3±1.8 12 ±1.6

6 Platelet count (103/mm3) 232.4±67.6 219.1±52.6 238.8±69.7
Serum cre atinine (mg/dl) 0.99 ±0.23 1.07±0.29 1.02±0.34
Total cholesterol (mg/dl) 134.1±26.2 215.2±48.1*† 165.7±31.4*
Triglyceride (mg/dl) 93.2±20.9 172.2±90.6*† 103.1±25.3
HDL -c (mg/dl) 40.2±3 33.4±4*† 36.6±4.1*
LDL -c (mg/dl) 75.3±26.5 146.4±39.5*† 108.5±3 1.4*
AMI: Acute myocardial infarction; St. CAD: Stable coronary artery
disease; BMI: Body mass index; HDL -c: High density lipoprotein;
LDL -c: Low density lipoprotein; AST: Aspartate aminotransferase; *:
significant versus control group; †: significant ver sus St. CAD
patients' group.

Table (2): Test validity characters of various cutoff points of
miRNA499 relative level as predictor of cardiac disease severity as
judged by Gensini angiographic scoring

11.2 RU 12.1 RU 12.5 RU 12.9 RU 13.1RU
Sensitivity Value 75% 80% 53.33% 20% 6.67%
95% CI 51-91% 52-96% 27-78.7% 4.3-48.1% 0.17-32%
Specificity Value 100% 100% 100% 100% 100%
95% CI 78-100% 83.16 -100%
PPV Value 100% 100% 100% 100% 100%
NPV Value 75% 86.96% 74.07% 62.5% 58.82%
95% CI 58-86.5% 71-94.8% 62.5-83% 56.4-68% 55.5-62%
Disease
prevalence Value 57.14% 42.86% 42.86% 42.86% 42.86%
95% CI 39-73.7% 26.32 -60.65%
AUC Value 0.217 0.773 0.682 0.528 0.971
P value =0.045 =0.021 =0.039 >0.05 =0.009
95% CI 0.063 -0.37 0.25-0.8 0.63-0915 0.5-0.866 0.91-1.03
RU: Relative unit; CI: Confidence interval; PPV: Positive predictive
value; NPV: Negative predictive value; AUC: Area under curve

7
Fig. (1): Mean value of relative miRNA -499 unit levels estimated in studied
patients and controls9.51010.51111.51212.51313.5Relative miRNA -499 UnitControl AMI CAD

Fig. (2): The relative miRNA -499 quantity (RQ) level was increased
by 3.187 and 2.202 fold among AMI p atients than controls and St.
CAD patients and by 1.447 fold in St. CAD patients than controls.

8
1 – Specificity1.0 0.8 0.6 0.4 0.2 0.0Sensitivity1.0
0.8
0.6
0.4
0.2
0.0
Fig. (3 ): ROC curve analysis for miRNA499 relative level at cutoff
point of 11.2 RU as predictor for Gensini angiographic score
1 – Specificity1.0 0.8 0.6 0.4 0.2 0.0Sensitivity1.0
0.8
0.6
0.4
0.2
0.0

Fig. (4 ): ROC curve analysi s for miRNA499 relative level at cutoff
point of 12.1 RU as predictor for Gensini angiographic score

Discussion
Diagnosis of severity and extent of coronary vascular
occlusion in studied cardiac patients relied on angiographic evaluation
of coronary vasc ulature interpreted using Gensini scoring system,
which reflects both severity and extent of coronary artery disease
(CAD). In support of the reliance on Gensini angiographic score as a

9 gold standard for evaluation of study outcome, multiple studies
applie d it for evaluation of the extent and severity of coronary
atherosclerosis in various disease states (Kruszelnicka et al., 2013,
Erkan et al., 2015, Pothineni et al., 2015) .
Estimated circulating level of miRNA -499 was significantly
higher in AMI patient s than controls and St. CAD patients and relative
miRNA -499 quantity level was increased among AMI patients by
3.187 and 2.202 fold than controls and St. CAD patients, while was
increased by 1.447 fold in St. CAD patients than control subjects.
In line with these results, Xiao et al., (2014) experimentally,
found serum miR -499 and miR -208a levels were extensively elevated
4-hr after induction AMI and the increase was sustained till 24 -hr
thereafter and also reported that miR -499 might be released from
damaged heart to the circulation.
Clinically, Ji et al., (2015) found the level of miR -133a, miR –
133b and miR -499-5p were significantly higher in both ST and non –
ST segment elevation MI patients compared to healthy volunteers.
Devaux et al., (2015) reported that in AMI patients, the levels of miR –
208b, miR -499 and miR -320a were significantly than patie nts with
other diagnoses. Also, Zhao et al., (2015) found the expression of
microRNA -499 in serum of patients with AMI was significantly
higher than in controls . Recently, Xin et al., (2016) documented that
miRNA -499 is mainly expressed in myocardium and its circulating
levels were increased in AMI patients
Estimated levels of circulating miRNA499 showed positive
significant correlation with Gensini angiography s core, serum
positivity for cTnI, serum CK -MB level and serum total cholesterol.
Similarly, Chen et al., (2015) found plasma miR -499 levels in AMI
patients were positively -correlated with cTnI, CK -MB, number of
affected coronary vessels and with Gensini sco res. Moreover,
Pourrajab et al., (2016) reported a significant positive correlation
between cTnI and miR -499 levels in patients undergoing cardiac
revascularization surgery.
Statistical analysis showed that circulating miRNA499 level at
range of 11.2 -12.1 RU could define high Gensini score with
specificity and PPV of 100%, sensitivity in range of 75 -80% and
disease prevalence rate ranging betwe en 42.9 and 57.1 %. In hand with
these findings, Cheng et al., (2014) documented that miRNAs,
especially miR -499 may be suitable for use as diagnostic biomarkers
of MI. Thereafter, Zhao et al., (2015) reported that the sensitivity and
specificity of microRNA -499 in judging AMI were 86.37% and

10 93.47%, respectively. Recently, Shalaby et al., (2016) found that the
AUC of m iRNA -499 and 210 for the diagnosis of acute coronary
syndrome (ACS) patients with symptoms onset <3 h were 0.89 and
0.86, respectively and concluded that both miRNAs could help to
accelerate the diagnosis of ACS patients in emergency unit.
ROC curve analys is defined circulating miRNA499 level at
cutoff points of 11.2 and 12.1 RU as sensitive and specific predictor of
high Gensini angiographic score, respectively. These findings go in
hand with Zhao et al., (2015) who documented that the high
sensitivity and specificity of miRNA -499 for AMI diagnosis suggest
that it would be useful as an auxiliary index for clinical diagnosis of
AMI. Also, Liu et al., (2015) confirmed the predictive values of
plasma miR -1, miR -208 and miR -499 in AMI and supposed that the
cardiac-specific miR -208 and miR -499 were more reliable as
biomarkers in AMI screening and prediction than miR -1. Furthermore,
Chen et al., (2015) concluded that cardiac -specific miRNA -499 might
prove to be a predictor of the risk of myocardial ischemia. Recen tly,
Pourrajab et al., (2016) reported that in contrast to conventional
biomarkers cTnI and CK, circulating levels of miRNAs decreased
significantly after revascularization surgery and could be used as early
biomarker which indicates myocardial ischemia in cardiac surgery.
Mean of time lapsed since onset of AMI attack and blood
sampling at emergency department was about 80 minutes, so
estimation of circulating miRNA499 level could be considered as
early predictor for both severity and extent of CAD as judge d by
Gensini angiographic score. In line with these findings, Ji et al.,
(2015) documented that level of miRNAs were considerably increased
within the first 4 hours, of both ST and non -ST segment elevation MI
patients. Also, Zhao et al., (2015) and Chen et al., (2015) found that
microRNA -499 was detected in serum 3 h post -AMI, reaching a peak
after 12 h and declining after 15 h.

Conclusion
Circulating levels of miRNA -499 could differentiate between
patients with AMI and stable coronary artery disease. Est imated
miRNA499 at range of 11.2 -12.1 RU showed high diagnostic test
validity characters and being specific for diagnosis of AMI, so
defining an acceptable range for prediction of the severity of cardiac
insult as preliminary early non -invasive diagnostic modality till
patient's condition allows angiographic evaluation.

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14 الممخص العربي
دراسة الحمض النووي الريبوزي الدقيق- 944 كداللة جزيئية الحتشاء عضمة القمب الحاد
لدي اإلنسان
أمــــــال ابو الفضل حسن عمي، أسامة سند عرفة *، عادل فرج الخولي، نجــالء فتحـى
ابراىيم الحسيني، شيماء دمحم عبد الرحمن بغدادى
أقسام الكيمياء الحيوية الطبية، أمراض القمب * – كمية الطب، جامعة بنيا

اليــد مـن البحــثي تقيـيم مــا اا كـان التعبيـر الجينــي لمحمـض النــووي الريبـوزي الــدقيق-
944 .مفيد كداللة جزيئية إلصابة عضمة القمب لدى اإلنسان
المرضـي وطرق البحثي شممت الدراسة 51 مريضـا مصـابا باحتشـاء عضـمة القمـب
الحــاد (مجموعـــة االحتشـــاء الحــاد ، 02 مريضـــا مصـــابا بــشمراض الشـــرايين التاجيـــة يـــر
احتشاء عضمة القمب الحاد(مجموعة أمراض الشرايين التاجية ، 51 متطوعا من األصـحاء
(المجموعــة الضــابطة . وخضــعت مجموعــات المرضــي لقســطرة والتصــوير الممــون لشــرايين
القمــب وتقيــيم النتــائ تبعــا لمقيــاس جينســن، فحــص مصــل الــدم لتحديــد م شــرات احتشــاء
عضــمة القمــب و لمتحميــل الكمــي لمتعبيــر الجينــي لنحمــاض النوويــة الريبوزيــة الدقيقــة عــن
طريق التفاعل المتسمسل لمبممرة العكسي باستخدام بوادئ خاصة.
النتــائ ي كــان معــدل م شــرات احتشــاء عضــمة القمــب بمصــل الــدم أعمــي بفــارق او داللــة
احصائية عند مرضي احتشاء عضمة القمب الحاد مقارنـة بـالمجموعتين ارخـريين، ووجـدت
زيـــادة اات داللـــة احصـــائية فـــي مســـتوي الحمـــض النـــووي الريبـــوزي الـــدقيق- 944 بـــين
مجموعة احتشاء عضمة القمب الحاد مقارنة بالمجموعتين ارخريين. وجدت عالقـة ايجابيـة
اات داللــة احصــائية بــين مســتوي الحمــض النــووي الريبــو زي الــدقيق-944 وبــين مقيــاس
جينســـن لمـــدي انســـداد شـــرايين القمـــب، كمـــا وجـــد أن مســـتوي الحمـــض النـــووي الريبـــوزي
الـدقيق- 944 الـاي يتـراوح بـين55.0 و50.5 وحــدة نسـبية يمكنـو التنبـ المبكـر لمــدي
انسداد شرايين القمب بمعدل حساسية 51-02 %، ومعدل خصوصية522 .%
الخالصةي قياسمستوي الحم ض النـووي الريبـوزي الـدقيق- 944 يمكـن مـن التمييـز بـين
احتشاء عضمة القمـب الحـاد و أمـراض الشـرايين التاجيـة يـر احتشـاء عضـمة القمـب الحـاد،
وعندما يتراوح مستواه بين 55.0 و50.5 وحدة نسبية يمكنوالتنبـ المبكـر لمـدي انسـداد
شرايين القمب بمعدل حساسية 51-02 %، ومعدل خصوصية522.%