Clinical Significance of Downregulated Protein Kinase C Iota (Prkci) Gene in Peripheral Leukocytes of Patients with Acute Myocardial Infarction

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* 1Postgraduated 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 protein 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 selected as the control group. The clinical data of all study subjects were recorded. 6 ml of venous blood was sampled from both two groups 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 molecular 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 stenosis <50% were divided into the con- impact on AMI(17). Meanwhile, some genes were trol group. All the study subjects were excluded found upregulated in the AMI patients with ST-seg- other diseases such as cancer, liver dysfunction, ment elevation, and this could be used as an adjunct renal dysfunction, peripheral arterial diseases, or means for the STMI assessment(18). More and more thyroid diseases. This study was conducted in evidence has proven that the gene expression levels accordance with the declaration of Helsinki. This in AMI patients were abnormal, and these abnor- study was conducted with approval from the Ethics mally expressed genes might be used as the molec- Committee of Jilin University. Written informed ular markers for the AMI diagnosis or the therapeu- consent was obtained from all participants. tic targets. However, different ethnic groups have different gene expression profiles, so it would be Peripheral lymphocytes sampling vital to obtain the specific genes from the diseased 6 ml of fasting peripheral blood was sampled race(s). from each study subject in the morning, and then In our previous study, we found the expres- added into EDTA-anticoagulated tube and stored at sions of some genes in the peripheral leukocytes of 4°C. The lymphocytes were also sampled with 4h AMI patients in Han nationality in northeast China of blood sampling. The peripheral lymphocytes changed significantly than those non-AMI were then obtained referring to the instructions of patients(19), among which the protein kinase C iota human peripheral blood lymphocyte separation (PRKCI) gene was significantly downregulated solution (Tianjin Haoyang Biological Manufacture (P=0.003517699, LogFC = -1.563554087). CO., LTD, Tianjin). The peripheral lymphocytes of Furthermore, according to the pathway analy- each subject were divided into two parts, with one sis, this gene was involved in multiple metabolic part (4 ml) for the total RNA extraction and the pathways such as insulin metabolism, platelet acti- other part (2 ml) for the total lymphocytic protein vation, intercellular connections, and wnt signaling extraction. pathway, and these factors were related with the occurrence of multiple AMI risk factors. In this Detection by reverse transcriptionpolymerase study, the expressions of PRKCI in AMI patients chain reaction (RT-PCR) were studied at the RNA and protein levels, respec- The total RNA was extracted from the tively, and the functions of this gene was also ana- acquired peripheral lymphocytes according to the lyzed, aiming to find the molecular marker(s) of kit instructions of total RNA extraction kit (Tiangen AMI and apply it (them) for targeting clinical treat- BioTechnology Co., Ltd. Beijing), and then detect- ment. ed the quality and concentration using agarose gel electrophoresis and spectrophotometer. The total Materials and methods RNA reverse-transcription was then performed according to the kit instructions of reverse tran- General data scription kit (TOYOBO, Shanghai); the obtained 20 AMI patients hospitalized and treated in the cDNA was then stored at -80°C for the next fluores- Department of Cardiology, China-Japan Union cence quantitative PCR. Clinical 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 software in Mx3005P. The PCR primer sequences were Clinical info AMI group(n=20) Control(n=20) P 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’- Total PRKCI CTTTGCAGTGAGGTTC- 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’ Table 1: RT-PCR primer sequences which were used in LDL-ch(mmol/L) 3.49±0.96 2.84±0.57 0.044 RT-PCR. RT-PCR: reverse transcriptionpolymerase chain reaction HDL-ch(mmol/L) 0.85±0.15 1.14±0.36 0.004

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- total protein obtained was detected using classic protein; AMI: acute myocardial infarction; Note: *, P<0.05. Western Blot assay, with β-actin as the reference RT-PCR results gene and PRKCI as the candidate gene. The prima- In this study, the solubility curves of the candi- ry antibodies were rabbit anti-mouse β-actin anti- date gene and the internal reference were both sin- body and rabbit anti-mouse PRKCI antibody, and gle-peak form, indicating the amplification prod- the secondary antibody was goat anti-rabbit IgG ucts had higher specificity (Figure 1), and no non- antibody. All the antibodies were purchased from specific amplification occurred. Abcam Co. The Δcycle threshold (ΔCT) value of each RT-PCR sample was the average of three-time repe- Statistical analysis titions, and the standard deviations were all in the All the data were analyzed using SPSS20.0 reasonable ranges. The relative expression of (Statistical Product and Service Solutions20.0, PRKCI used the 2-ΔΔCt method, and the results IBM, USA) statistical software; all the measure- showed that at the mRNA level, the expression of ment data were expressed as mean±standard devia- PRKCI in the AMI group was lower than the con- tion, and the intergroup differences were performed trol group, and the relative expression of was 0.62 ± the independent T test; the counting data were 0.11, P=0.032 (Table 3), consistent with the results expressed as frequency, and the intergroup compar- of the microarray assay(19). ison used the χ2 analysis; with P<0.05 considered as statistically significant. 316 Lin Fan, Heyu Meng et Al

this study confirmed that PRKCI was downregulated 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 biological processes as muscle contraction, gene expression, 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; (23- B: Solubility curve of candidate PRKCI. such as cardiac arrhythmia or cardiac dysfunction 25) RT-PCR: reverse transcriptionpolymerase chain reaction . 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 P<0.05. Encyclopedia (http://www.kegg.jp/pathway/ Western blot hsa04910) showed that the combination of insulin The results of Western blot showed no signifi- and insulin receptor could then activate PIP3 and cant difference in the expression of β-actin between proline-directed protein kinase 1 (PDPK1), whose the two groups, but the protein expression of activation would further activate the PRKCI pro- PRKCI in the AMI group was significantly lower tein. The activated PRKCI protein then indirectly than the control group (Figure 2). activated the sterol regulatory element binding protein 1 (SREBP-1C) and fatty acid synthase (FAS), thus promoting adipogenesis indirectly; meanwhile, it could promote the absorption of acetylcoenzyme a carboxylase α and glucose. In addition, PDPK1 could cause the threonine Fig. 2: Results of Western blot. protein kinase 3 (Akt3) phosphorylated, and the lat- The results show that the internal reference gene GAPDH had the same expressions in the two groups, and the PRKCI gene in ter could inhibit the fat metabolism by inhibiting Group AMI was downregulatedthan Group CON. the synthesis of cAMP. On the other hand, the acti- 1-3: Samples of the control group; 4-6: Samples of the AMI vated SREBP-1C could indirectly activate the pyru- group. AMI: acute myocardial infarction vate kinase (PYK) and glucokinase (GK), and these RT-PCR: reverse transcriptionpolymerase chain reaction two enzymes could reduce the blood sugar via the glycolytic pathway. Discussion In the platelet activation pathway, the combination of ADP and G protein-coupled purine P2Y Our previous microarray experiments revealed receptor 1 (P2Y1) on the platelet membrane would that the PRKCI gene in the peripheral leukocytes of activate the G protein polypeptide (Gq), and the lat- AMI patients was significantly downregulated, and Clinical 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 interference 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-atherosclerosis. 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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: [email protected] (China)