CLINICAL SIGNIFICANCE OF DOWNREGULATED PROTEIN KINASE C IOTA (PRKCI) GENE IN PERIPHERAL LEUKOCYTES OF PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
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Acta Medica Mediterranea, 2018, 34: 313
CLINICAL SIGNIFICANCE OF DOWNREGULATED PROTEIN KINASE C IOTA (PRKCI) GENE IN
PERIPHERAL LEUKOCYTES OF PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
LIN FAN1, HEYU MENG2, YUSHUANG YANG3, KAIYAO SHI3, DONGNA LIU3, FANBO MENG3*
1
Postgraduated student of Department of Cardiovascular Medicine, China-Japan Union Hospital of Jilin University, now work in
Echocardiography Department, First Affiliated Hospital of Soochow University, Suzhou 215006, China - 2Medical College of Yanbian
University, Yanji 133000, China - 3Department of Cardiovascular Medicine, China-Japan Union Hospital of Jilin University, Jilin
130033, China
ABSTRACT
Introduction: It has been accepted that gene play a role in the evolution of acute myocardial infarction (AMI). We found pro-
tein kinase C iota (PRKCI) gene had different expression in the peripheral leukocytes of patients with AMI through gene chip.
Materials and methods: 20 AMI patients were randomly selected and set as the AMI group, and 20 healthy people were selec-
ted as the control group. The clinical data of all study subjects were recorded. 6 ml of venous blood was sampled from both two grou-
ps for ribonucleic acid (RNA) and protein extraction; reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot
were then performed to detect the expression of PRKCI gene using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-
actin as the internal reference, respectively.
Results: There existed significant differences in low-density lipoprotein (LDL-ch) and high-density lipoprotein (HDL-ch)
between the two groups. At the RNA level, the relative expression of PRKCI gene in the AMI group to the control group was 0.62 ±
0.11 (P=0.032); at the protein level, the PRKCI protein expression in the AMI group was also significantly reduced than the control
group.
Conclusions: The low expression of PRKCI gene takes part in the development of AMI. PRKCI gene may be used as a molecu-
lar marker for the diagnosis of AMI, even treated as a therapeutic target for the clinical diagnosis and treatment of AMI patients.
Keywords: Acute myocardial infarction, PRKCI gene, RNA, PRKCI protein, molecular labeling.
DOI: 10.19193/0393-6384_2018_2_50
Received August 30, 2017; Accepted January 20, 2018
Introduction Present treatment methods such as anti-platelet
aggregation, coronary artery expansion, blood fat
Acute myocardial infarction (AMI) refers to reduction, or coronary stent implantation reduce the
the coronary artery stenosis or occlusion-based morbidity and mortality of AMI to some extent, and
drastic reduction or interruption of coronary blood they also play important roles in the prognosis and
supply, thus causing severe ischemia-resulted recovery of these patients.
myocardial necrosis in the corresponding myocardi- However, partial patients would still occur
um. Studies in recent years have shown that AMI is secondary AMI, and the treatment effects would
a disease co-caused by a variety of factors such as thus be poor. Studies found that some patients still
heredity, environment, personal habits, or society, occur thrombosis when orally administrated clopi-
and it also has the characteristic of familial aggre- dogrel, and it was related with the mutation of cer-
gation; however, its pathogenesis could not be fully tain gene locus(2-4). Therefore, selecting one optimal
explained by the Rules of Mendelian inheritance. drug treatment through clinically testing these
Therefore, it is now considered as a polygenic patients’ gene sequences could greatly increase the
inheritance disease(1). treatment effects.314 Lin Fan, Heyu Meng et Al
In recent years, microarray technologies have Hospital of Jilin University, from November 2014
been successfully applied to investigate the genetic to January 2015 were randomly selected and set as
correlations of various diseases, including hyper- the AMI group. In the meantime, 20 healthy volun-
tension(5), type 1 diabetes(6), or pancreatic cancer(7). teers were set as the control group. The clinical data
The whole genome association studies have shown of these two groups were recorded and analyzed in
that AMI is related with heredity, and the locus details. The diagnostic criteria of AMI referred to
mutations of some genes are closely related with the diagnostic guidelines issued by the American
the occurrence of AMI(8-11). Related researches also College of Cardiology/American Heart Association
made great progresses (12-14) , and PCSK9 gene in 2012(20), and the vascular lesions were further
inhibitors that are closely related with lipid metabo- confirmed by coronary angiography.
lism have been used in clinical treatments(15,16). The study subjects that met the diagnostic
At the RNA level, certain research also found guidelines and existed severe vascular stenosis or
that when compared with the sham group, the occlusion were set as the AMI group, while those
myocardial cells in the AMI mice exhibited some mismatching the diagnostic guidelines and with
gene expression differences, and this might have an vascular stenosisClinical significance of downregulated protein kinase C Iota (PRKCI) gene in peripheral leukocytes ... 315
The obtained cDNA sample was firstly diluted Results
by 10 folds, and then performed PCR amplification
according to the specific instructions of SYBR fluo- Baseline analysis
rescence quantification kit (TaKaRa, Dalian). The The baseline data of the study subjects were
reaction condition was at 95°C for 10 min, and the shown in Table 2. There was no significant differ-
following three steps were repeated 40 cycles: 95°C ence in the age, sex, and comorbidities between the
for 15 sec, 60°C for 20 sec, 72°C for 20sec; 95°C two groups; however, there were significant differ-
for 1 min, 55°C for 30 sec, and 95°C for 30 sec. ences in low-density lipoprotein (LDL-ch) and
The GAPDH gene was used as the reference gene, high-density lipoprotein (HDL-ch) between the two
and the PRKCI gene was set as the candidate gene; groups, and the AMI group exhibited higher LDL-
the specific amplification conditions were deter- ch level and lower HDL-ch level.
mined according to the solubility curves of the soft-
Clinical info AMI group(n=20) Control(n=20) P
ware in Mx3005P. The PCR primer sequences were
shown in Table 1. Age(years) 51±13 58±11 0.13
Gene Primer sequence Gender(F/M) 6/4 13-Jul 1
Upstream primer 5’-
GAPDH GACATCAA- Hypertension(yes/no) 8/12 14-Jun 0.74
GAAGGTGGTGAAGC -3’
Diabetes (yes/no) 5/15 18-Feb 0.405
downstream primer 5’-
TGTCATTGAGAG-
Fasting blood
CAATGCCAGC -3’ 7.16±2.63 6.04±3.51 0.306
sugar(mmol/L)
Upstream primer 5’-
PRKCI CTTTGCAGTGAGGTTC- Total
4.94±1.18 4.54±0.79 0.311
GAGAT - 3’ cholesterol(mmol/L)
downstream primer 5’-
AGCCTCTTCTAACTC- Triglyceride(mmol/L) 2.11±0.92 1.66±1.22 0.303
CAACTGAG -3’
LDL-ch(mmol/L) 3.49±0.96 2.84±0.57 0.044
Table 1: RT-PCR primer sequences which were used in
RT-PCR. RT-PCR: reverse transcriptionpolymerase chain
HDL-ch(mmol/L) 0.85±0.15 1.14±0.36 0.004
reaction
Western blot Blood platelet
242.35±63.92 234.92±44.75 0.724
The total protein was extracted from the count(10^9/L)
acquired peripheral lymphocytes using RIPA lysate, Table 2: Baseline information of the study subjects:
and its concentration was then detected using a there were no significant differences between the two
spectrophotometer; the obtained sample was then group except LDL-ch and HDL-ch.
stored at -80°C for the further Western Blot. The LDL-ch: low-density lipoprotein; HDL-ch: high-density lipo-
protein; AMI: acute myocardial infarction; Note: *, P316 Lin Fan, Heyu Meng et Al
this study confirmed that PRKCI was downregulat-
ed in Han AMI patients in northeast China through
more samples, no matter at the RNA level nor at the
protein level, this gene was detected significantly
downregulated.
The PRKCI gene is one member of the protein
kinase C family, which participates in a variety of
cellular responses, and whose activation plays
important roles in the early stage of various biologi-
cal processes as muscle contraction, gene expres-
sion, or cell proliferation(21,22). In myocardial tissues,
the protein kinase C is considered to be able to acti-
Fig. 1: Solubility curves of RT-PCR. vate the K-ATP channel, thus impacting the adap-
The results indicate that the primers were designed well, and tive regulation in the early ischemic stage; mean-
theamplified products had high specificity; the results of RT- while, it also participates in the occurrence of a
PCR had good reproducibility and were reliable. variety of post-myocardial ischemia complications,
A: Solubility curve of internal reference GAPDH;
B: Solubility curve of candidate PRKCI.
such as cardiac arrhythmia or cardiac dysfunction(23-
RT-PCR: reverse transcriptionpolymerase chain reaction
25)
. The PRKCI gene was found to be involved in
the insulin-mediated glucose transportation(26,27). It
was found in the extensive ischemic myocardial tis-
Group Average ΔCT ΔΔCT 2-ΔΔCt P
sues of rats that the PRKCI protein was decom-
AMI 10.99±0.84 posed into fine granular fragments and then trans-
0.70±0.31 0.62±0.11 0.032*
Control 10.29±0.89 ported, and showed no significant change after
reperfused with 1μM phorbol ester(28). Recent stud-
Table 3: CT values of each RT-PCR sample: the relative
expression of PRKCI in AMI group was 0.62. ies showed that PRKCI was related with the occur-
ΔΔCT = ΔCT of AMI group - ΔCT of control group; the CT rence of a variety of cancers, so it was considered
value referred to the number of the cycles (unit: times). as an oncogene(29).
AMI: acute myocardial infarction; RT-PCR: reverse transcrip- In the insulin pathway, the pathway analysis
tionpolymerase chain reaction; CT: cycle threshold; Note: *, on the website of the Japanese genes and Genomes
PClinical significance of downregulated protein kinase C Iota (PRKCI) gene in peripheral leukocytes ... 317
ter could activate the phospholipase C-β1 (PLCβ) platelet functions, so this phenomenon still needed
and thus further activate diacylglycerol (DAG), further study.
thereby activating the PRKCI protein. The activated Through analyzing the results in our study, we
PRKCI protein then could activate the Ras related considered that: such biological processes as the
protein RAP-1a, and then continuously induce the lipid metabolism, lipid synthesis, carbohydrate
activation of RIAM protein and talin 1 protein, metabolism, carbohydrate synthesis, and platelet
thereby causing the activation of the platelet mem- aggregation in human were subject to the regulation
brane integrin α-2b, which was also a component of of a variety of factors, so it would not be accurate
the platelet protein IIb/IIIa complex, so the activat- to analyze the results from only one single gene or
ed integrin α-2b could activate the fibrinogen α one metabolic pathway, so there would exist differ-
chain, thus further promoting the platelet aggrega- ences with the actual situations. AMI is a polygenic
tion indirectly. disease caused by the combination of multiple fac-
The pathway analysis showed that the normal tors, and lots of pathways are involved in, so the
expression of the PRKCI protein could be prone to interactions among these pathways would be com-
promoting the adipogenesis, blood glucose degra- plex, and more clinical data and gene function data
dation, and platelet aggregation, so the crowd with would help to improve the accuracy of further
downregulated PRKCI protein should have lower detailed analysis.
blood lipid levels and higher blood sugar levels. In recent years, individualized treatment of
However, it is generally believed AMI patients have certain disease has been gradually paid high atten-
higher LDL-ch lever, which is also one of the initi- tion in the medical field, and the targeting individu-
ating links of coronary atherosclerosis, contradict- alized treatment would greatly improve the effec-
ing this pathway analysis. In our previous cell tiveness of the treatment. Based on the results of
experiments, we used RNA interference vectors to previous microarray experiments, this study used
specifically interfere the in vitro rat cardiomy- blood samples, which could be easily obtained clin-
ocytes, and the non-interfered cells were used as a ically, to analyze the PRKCI gene expression in the
control so as to avoid the reduce the RNA gene and AMI patients, and the results revealed that the
protein expressions of PRKCI in the experiment expression of this gene was reduced in the AMI
group while the expressions of other genes were not patients, which thus promoted the synthesis of
affected. The comparative observation with the con- LDL-ch and thus induced the coronary atheroscle-
trol group revealed that the LDL concentration in rosis, so it could be considered as one of the causes
the media of the experiment group was gradually of AMI. Therefore, the PRKCI gene could be used
increased over time(30), indicating that the downreg- as a molecular marker for the diagnosis of AMI, or
ulation of the PRKCI gene could promote the syn- a therapeutic target for the clinical diagnosis and
thesis of blood lipids. treatment of AMI patients.
In the present study, the LDL-ch level in AMI
patients was higher than the control group, consis- Conclusions
tent with the results of our previous cell interfer-
ence experiments and the large clinical big data. The PRKCI gene was downregulated in the
The result that the HDL-ch level in AMI patients peripheral leukocytes in AMI patients, and its
was lower was also consistent with the general impacts on the lipid metabolism was one of the
belief that this factor was one protective media, and causes of AMI.
it could do favor to the formation of anti-athero-
sclerosis. According to the pathway analysis, the
patients with downregulated PRKCI gene should
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Acknowledgments _________
This study was funded by the Excellent Talent Projects in New Corresponding Author:
Century of Ministry of Education (2008), Projects of Jilin FANBO MENG
Provincial Science and Technology Department in 2009 Department of Cardiovasular Medicine,
(20090734), and Projects of Jilin Provincial Department of China-Japan Union Hospital of Jilin University,
Finance in 2012 (2012009), and special acknowledgements No. 126 Xiantai Street
should be given to Professor Zhihui Zhao and his research Changchun 130033
team for their technical guidance. E-mail: docfanbomeng@126.com
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