- Email: [email protected]
Long Noncoding RNA AK123483 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3
HLC 2382 1–8
ORIGINAL ARTICLE
Heart, Lung and Circulation (2017) xx, 1–8 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2017.04.011
Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3
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Chengfei Zheng, Lu Tian, Donglin Li, Ziheng Wu, Xiaohui Wang, Yunjun He, Yangyan He, Wei Jin, Ming Li, Qianqian Zhu, Tao Shang, Hongkun Zhang *
Q2 The First Affiliated Hospital of Zhejiang Medical University, China Received 22 January 2017; received in revised form 11 April 2017; accepted 15 April 2017; online published-ahead-of-print xxx
[1_TD$IF]Backgroud
Recently long non-coding RNAs (lncRNAs) have attracted attention in several biomedical fields. The purpose of this study is to investigate the profile of myocardial lncRNAs and their potential roles in myocardial ischaemia-reperfusion injury (IRI).
Methods
EdgeR bioconductor package was used to screen differentially expressed lncRNAs in myocardial IRI, and lncRNA AK12348 was selected. The mRNA levels of lncRNA AK12348 in normal and anoxia/reoxygenation (A/R) cardiomyocytes were determined by qRT-PCR. After transfection with siRNA-lncRNA, AK12348, LDH release and cell apoptotic rates in normal and A/R cardiomyocytes were determined. The protein expression values of PARP and Caspase-3 were also determined by western blotting.
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Results
The relative level of lncRNA AK12348, LDH release and cell apoptotic rate in A/R cardiomyocytes was significantly higher than that in normal cardiomyocytes. After transfection with siRNA-lncRNA AK12348, LDH release and cell apoptotic rates in A/R cardiomyocytes were reduced, while the values in normal cardiomyocytes had almost no change. The protein expression values of PARP and Caspase-3 in A/R cardiomyocytes were much higher than the Control. After knockdown of lncRNA AK12348, the values decreased.
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Conclusion
Long non-coding RNAs AK12348 could be potential therapeutic targets for the treatment of myocardial IRI.
Keywords
LncRNA AK12348 Myocardial IRI A/R LDH PARP
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Introduction Q12
Coronary heart disease (CHD) is the leading cause of death and disability in the world. According to the WHO, 7,254,000 deaths worldwide (12.8% of all deaths) resulted from CHD in 2008. The effects of CHD are usually caused by acute myocardial ischaemia-reperfusion injury (IRI) [1]. Ischaemiareperfusion injury is a complex phenomenon involving many
players, all contributing to the final damage inflicted on the heart [2]. Ischaemia-reperfusion causes damage to membrane phospholipids, and is a significant mechanism of cardiac I/R injury. Molecular dissection of sarcolemmal damage in I/R, however, has been difficult to address experimentally [3]. Long non-coding RNAs are RNAs that are longer than 200 nucleotides in length and do not code for proteins, although
*Corresponding author. Email: [email protected] © 2017 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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they can interact with proteins [4]. They are involved in numerous biological roles including epigenetic regulation, apoptosis, and cell cycle [5]. Emerging evidence suggests that lncRNAs may serve as master gene regulators capable of controlling protein-coding and non-coding genes and, as such, they have been implicated in the regulation of a variety of cellular functions and disease processes including stemness and cancer metastasis [6,7]. A study by Liu et al. showed gene ontology and pathway analysis indicated lncRNAs play important roles in the post-ischaemic heart [8]. However, further exploration of the effect of lncRNAs on myocardial IRI has not been conducted. In this study, we screened the differentially expressed lncRNAs in primary cardiomyocytes with myocardial ischaemia-reperfusion injury. The effect of lncRNA on the proliferation of cardiomyocytes was studied and the molecular mechanism was explored.
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Material and Methods
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Identification of Differentially Expressed lncRNA
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The microarray expression profiles of GSE50378 and GSE22282 were downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database. Gene expression profiles between them were compared. We transferred the probe-level data in CEL files into expression measures. Then, background was corrected and quartile data was normalised by the robust multiarray average (RMA) algorithm. The file in the platform annotation files provided by Affymetrix Company was used to map the relationship between the probes and gene symbols. A probe would be filtered if the probe did not have the corresponding gene symbol. The average value of the gene symbol with multiple probes obtained would be further analysed. The primary comparison of samples from HS patients and healthy donors was performed by Linear Models for Microarray Data (LIMMA) package. A set of gene-specific t tests with the threshold of false discovery rate (FDR) 0.05 was assimilated to identify differentially expressed lncRNAs.
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Tissue Samples and Cell Culture
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Fifty-five pairs of tissues were collected from rats with myocardial ischaemia/reperfusion injury and normal rats. After isolation, the tissues were stored at 80 C. Rat cardiomyocyte H9C2 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were kept in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) in humidified air containing 5% CO2 at 37 C. Dulbecco’s Modified Eagle’s Medium contained 10% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin/ streptomycin.
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Cell Transfection
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RNA interference (RNAi) is an evolutionarily conserved surveillance mechanism that responds to double-stranded
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RNA by sequence-specific silencing of homologous genes. To investigate the role of lncRNA AK12348 on myocardial cells, we used a RNAi-based strategy (c-lncRNA AK12348-siRNA) to specifically silence lncRNA AK12348 expression. LncRNA AK12348-siRNA transfection (50 nmol/L) was performed in cultured myocardial cell H9C2. Then, qRT-PCR was used to detect relative mRNA level.
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RNA Isolation and Quantitative Realtime Reverse Transcription-PCR (qRTPCR)
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TRIzol reagent[12_TD$IF] (Invitrogen) was used to isolate total RNA from tissues or cells. Total RNA was used to synthesise cDNA with PrimeScript reverse transcription-PCR kit (TaKaRa, Shiga, Japan), and subjected to quantitative realtime PCR. The relative expression levels of miR-181a were measured using a SYBR PrimeScript miRNA quantitative real-time polymerase chain reaction Kit (TaKaRa, Shiga, Japan) following the manufacturer’s instructions, with U6 as an internal control. MiR-181a mRNA expression levels were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), with GADPH as an internal control. The reaction was performed on an ABI 7500 Sequence Detection System (ABI, Vernon, CA, USA).
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Western Blotting
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Total proteins from the cells were extracted by ice-cold RIPA lysis buffer supplemented with 1 mM proteinase inhibitor PMSF (Sigma, St. Louis, MO, USA). The protein concentration was quantified with a BCA assay kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF, Millipore, Bedford, MA, USA) membrane, and then blocked with 5% non-fat milk in Tris-buffered saline. The membranes were incubated with primary antibodies, mouse antihuman monoclonal ZEB1, E-cadherin, Vimentin and N-cadherin antibody (Santa Cruz Biotechnology, CA, USA) and mouse anti-human monoclonal b-acitin antibody (Santa Cruz Biotechnology, CA, USA), at 4 C overnight. The membranes were washed and subsequently probed with secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1/4000 dilution for 1 h at room temperature. Proteins were visualised with chemiluminescent detection system (ECL; Beyotime). GAPDH was used as internal control.
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Flow Cytometry Assay
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Flow cytometry assay was used to clarify cell apoptosis. Cells were collected with trypsinisation and then washed twice with PBS, and fixed in cold 80% ethanol, and finally stored at 4 C overnight. The cells were washed with PBS and RNase A was administered. Propidium iodide was added to tubes and then incubated for 20 min at 4 C in the dark. FITC-labelled Annexin V/PI staining was applied. 1 106 cells in each well were suspended with buffer containing FITC-conjugated
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Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Regulation of Myocardial Ischaemia-Reperfusion Injury
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Annexin V/PI. Samples were then analysed via flow cytometry.
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Immunohistochemistry
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Cell histologic sections were deparaffinised, rehydrated, and blocked with methanolic 3% hydrogen peroxide. The slides were immersed in 10 mm citrate buffer, pH 6.0 and heated in a 1200 W microwave oven at the highest power setting. Evaporated liquid was replenished at 5 mins, then the slides were heated at high power for an additional 5 mins. The slides were left in the buffer for an additional 10 mins before being removed. The antibody used was a mouse monoclonal antibody raised against full-length recombinant Cdx2 protein (AM392–5 m; BioGenex, San Ramon, CA). Immunostaining was performed by hand using the prediluted antibody solution and a 30-min incubation. After washing, antibody binding was visualised using the avidin-biotinperoxidase technique (Vectastain Elite ABC kit, Vector Laboratories, Burlington, VT), followed by incubation with [13_TD$IF]3,30 diaminobenzidine tetrahydrochloride. The slides were counterstained with haematoxylin.
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Statistical Analysis
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Statistical analyses were performed using SPSS Statistics (SPSS Inc., Chicago, IL, USA). All data were expressed as
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mean standard deviation. Student[14_TD$IF]’s t-test or one-way ANOVA test was performed to determine significant differences. A p ＜ 0.05 was considered statistically significant.
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Results
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Screening of Differentially Expressed lncRNA
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To explore the lncRNAs that play key roles in myocardial IRI, lncRNAs data were downloaded from the database and uploaded to GEO (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi[25_TD$IF]? acc=GSE50378; http://www.ncb i.nlm.nih. gov/geo/query/acc.cgi?acc[16_TD$IF]=GSE22282) to screen differentially expressed genes. Results showed 26 differentially expressed lncRNAs were taken as representative in GSE50378, of them, eight lncRNAs were up-regulated by more than 1.5-fold, and 18 miRNAs were down-regulated by more than 1.5-fold. In GSE22282, 65 differentially expressed lncRNAs were taken as representative. Of those, 30 were down-regulated and 35 were up-regulated ([1_TD$IF]Figure 1). The top 26 differentially expressed lncRNAs in GSE50378 are shown in Table 1a, the differentially expressed lncRNAs in GSE22282 are shown in Table 1b. There are 164 differentially expressed lncRNAs in GSE50378 and 119 differentially
[1_TD$IF]Figure 1 Heatmap of lncRNAs in myocardial ischaemia-reperfusion injury tissues that was significantly down-regulated or up-regulated. For each miRNA the red colour means an up-regulated expression, and the blue colour means a downregulated expression. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Table 1a Top 26 differentially expressed lncRNAs in GSE50378 samples. GSE50378
p Value
AK123483 AK024898
3.779478 2.202297
1.67E-08 1.43E-06
AL389942
1.743006
4.30E-06
BC004287
1.552815
6.80E-06
AK093009
1.744483
1.71E-05
AL110176
1.626017
2.26E-05
AK123079
1.341703
4.78E-05
AK091312
1.105875
6.17E-05
CR611122 BC013423
1.31421 1.095659
6.32E-05 7.52E-05
AK123657
1.00934
7.56E-05
AK023774 CR613326
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logFC
1.341952 1.13839
9.38E-05 9.82E-05
BC020900
1.050885
0.000108
AK021543
1.107317
0.000115
BC043185
1.047101
0.000126
AY605064 AK023526
1.10054 1.768026
0.000143 0.00022
AK055670
1.0225
0.000309
EU249757
1.02182
0.000324
AK058186
1.04111
0.000354
AJ227892
1.432
0.000376
BC041955
1.050115
0.000451
AK057923
1.292861
0.002547
BC043182 BC018139
1.286492 1.20351
0.007411 0.008528
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expressed lncRNAs in GSE22282. Among them, four lncRNAs were the same and they were AF495725, AK098081, AK025695 and BC023972 (Table 2).
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Myocardial Cell Identification
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To explore the effect of lncRNA on rat cardiomyocytes more precisely, we isolated cardiomyocytes to culture for the further study. Immunofluorescence assay was used to determine the purity of cultured primary cardiomyocytes. The immunofluorescence result of cardiac troponin T (cTnT) showed the cytoplasm of cardiomyocytes presented green fluorescence, which indicated immunofluorescence staining was positive ([1_TD$IF]Figure 2A). It indicated the purity of cultured primary cardiomyocytes was well and could be used for the next experiment. The mRNA levels of lncRNA AK123483 in normal primary cardiomyocytes (Control) and cells with anoxia/reoxygenation were determined by qRT-PCR. Results showed the level of lncRNA AK123483 in the A/R group was significantly higher than that in the Control ([1_TD$IF] Figure 2b).
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Cell Transfection
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We used the lentivirus pGFP-V-RS-lncRNA AK123483 siRNA and pGFP-V-RS vector to infect cardiomyocyte
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H9C2. The transfection efficiency was also determined by fluorescence microscope, and results showed most cells were transfected with pGFP-V-RS-lncRNA AK123483 siRNA in the silncRNA AK123483 groups ([1_TD$IF]Figure 3a). The mRNA levels of lncRNA AK123483 in the Control, si-Con, siRNA1, siRNA2 and siRNA3 groups were determined by qRT-PCR. Results showed the mRNA levels of lncRNA AK123483 in the siRNA1, siRNA2 and siRNA3 groups were significantly higher than that in the Con and siCon groups ([1_TD$IF]Figure 3b).
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Knockdown of lncRNA AK123483 Influenced LDH Release and Cell Apoptosis
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The lactate dehydrogenase-A (LDH-A) enzyme catalyses the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumour outcomes [9]. In this study, results showed knockdown of lncRNA AK123483 had no influence on LDH release in primary cardiomyocyte (Control); LDH release in A/R primary cardiomyocyte was much higher than that in the Control. After transfection with siRNA-lncRNA AK123483, LDH release decreased significantly ([1_TD$IF]Figure 4a). The influence of lncRNA AK123483 expression on the apoptosis of cardiomyocytes at A/R condition was also determined by flow cytometry. Results showed knockdown of lncRNA AK123483 had no influence on apoptotic rate in the Control group. The apoptotic rate in the A/R group was markedly higher than that in the Control. After A/R primary cardiomyocyte was transfected with siRNA-lncRNA AK123483, the apoptotic rate decreased dramatically ([1_TD$IF]Figure 4b and c).
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Knockdown of lncRNA AK123483 Regulated the Expression of PARP
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As the above results show, A/R could promote the apoptosis of primary cardiomyocytes, and knockdown of lncRNA AK123483 reduced the apoptotic rate. However, the molecular mechanism was unknown. The protein expression values of PARP and Caspase-3 in normal primary cardiomyocyte, and A/R primary cardiomyocyte before and after transfection with siRNA were determined by western blotting. Results show the protein expression values of PARP and Caspase-3 in the A/R group were much higher than the Control. After A/R primary cardiomyocyte was transfected with siRNA, the values decreased ([17_TD$IF]Figure 4b).
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Discussion
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Ischaemic heart disease is a major cause of death and often caused by coronary artery disease. Comprehensively deleted cardiomyocytes is related to ischaemia and reperfusion injury (IRI) [10]. Myocardial IRI could result in many inflammatory responses including production of oxidants, activation of complement, and infiltration by polymorphonuclear neutrophils [11]. Cardiomyocyte death/apoptosis plays an important role in ischaemic hearts. As reported, various genes are abnormally expressed in infarcted hearts, which
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Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Regulation of Myocardial Ischaemia-Reperfusion Injury
Table 1b Differentially expressed lncRNAs in GSE22282 samples. GSE22282
logFC
p Value
logFC
p Value
logFC
p Value
0.834103 1.43372
0.019611 0.019696
0.774723
0.020929
BC053563 BC024020
1.336855 1.638161
4.07E-05 4.64E-05
BC031342 AF318321
1.2318 1.100853
0.011321 0.011347
AK126861 AK098081
U94902
1.406273
9.22E-05
AF086441
1.363236
0.011508
AK291479
AY216265
2.255815
0.000743
AF052160
0.76307
0.011545
AK026440
0.43023
0.021097
0.001072
BC000986
0.50081
0.011714
BC037530
0.52414
0.021661
AK094088
3.80262
AF495725
1.116279
0.001094
AK002146
2.08507
0.011897
AK090808
0.76373
0.023217
BC014583
0.913022
0.001272
AB007954
0.63759
0.0125
AK054868
0.60838
0.023446
BC040834
2.073123
0.002196
AK024965
0.012593
BC039682
1.45356
0.024294
AF086543 AK057657
1.148865 0.64271
0.003023 0.003606
AF075045 AK054971
1.32901 0.66463
0.012735 0.013026
U72512 AK095104
0.54101 1.383514
0.024664 0.025161
BC006384
0.784642
0.003789
AX721088
0.56321
BC018655
0.831002
0.004952
AK021459
AK092600 AK025695
0.57829
0.005344
AF090921
0.826167
0.005639
AK025166
0.971376
0.813113
0.01349
AL049450
0.4768
0.025646
0.843602
0.013912
BC041399
0.3878
0.026269
0.978045
1.36545
0.013928
AK002088
0.62692
0.014248
CR595807
0.0144
0.006468
AK022196
1.31242
AK311445
0.57075
0.006541
BC035078
0.47204
AK024385 AK026372
2.45816 0.935024
0.006929 0.007245
AF147344 AK090404
BC039325
1.28851
U94903
AK021555 AK021643 BC037234
0.467489 1.29959 1.008319
0.02638 0.026814
0.015331
0.740554 0.70235
0.015621 0.015742
0.008109
uc003lfc.1
0.54317
0.015788
0.009064
uc004ebu.1
0.54809
0.0159
0.009288
AL109779
1.14044
0.017035
0.009724
AK021803
1.40662
0.017181
AK023330
1.373985
0.009752
AK123379
0.6377
0.017608
AB019562
2.045813
0.009763
AK022157
0.53792
0.018188
AK130460 AK091637
0.59566 0.960152
0.010953 0.010955
AK123861
0.811307
1.687597
0.018856
Figure 2 A. Immunofluorescence results of cultured primary cardiomyocyte. B. Expression level of lncRNA AK12348 in normal and A/R primary cardiomyocyte. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Table 2 Differentially expressed lncRNAs in both GSE50378 and GSE22282. logFC
GSE50378
GSE22282
p Value
Average expression
Average expression
level (normoxial)
level (hypoxial)
AF495725
0.529028
0.003148
8.53034
9.059368
[5_TD$IF]AK098081
0.64122
0.003381
9.751949
9.110731
[6_TD$IF]AK025695
0.72119
0.001259
9.699084
8.977898
[7_TD$IF]BC023972
0.78286
0.00049
4.09202
3.309155
AF495725
1.116279
0.001094
6.911135
8.027415
[8_TD$IF]AK098081
1.43372
0.019696
7.972042
6.538321
[9_TD$IF]AK025695
0.826167
0.005639
8.881449
9.707616
[10_TD$IF]BC023972
0.597165
0.030373
5.020953
5.618119
Figure 3 Knockdown of lncRNA AK12348 influenced LDH release. A. Normal and A/R primary cardiomyocyte were transfected with siRNA-lncRNA AK12348. The transfection efficiency was determined by fluorescence microscope. B. LDH release in normal and A/R primary cardiomyocyte before and after transfection with siRNA- lncRNA AK12348. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
HLC 2382 1–8
7
Regulation of Myocardial Ischaemia-Reperfusion Injury
Figure 4 Knockdown of lncRNA AK12348 influenced the apoptosis primary cardiomyocytes. A. The apoptosis situation of normal and A/R primary cardiomyocytes before and after transfection with siRNA-lncRNA AK12348. B. The protein expression values of PARP and Caspase-3 in normal and A/R primary cardiomyocytes before and after transfection with siRNA-lncRNA AK12348.
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can result in cardiac remodelling after IRI [12]. The cellular mechanisms underlying IRI are complex and involve many signalling pathways and molecular players [13]. Therefore, it is necessary to find multiple molecular targets. Since lncRNAs are found to regulate gene expression, it is reasonable to suppose that lncRNAs may play an important role in IRI. In this study, we screened the differentially expressed lncRNAs in GSE50378 and GSE22282, which were downloaded from GEO, and the top 26 differentially expressed genes were obtained. LncRNA AK12348 was the most differentially expressed gene in GSE50378 and selected for the further study. We obtained the relative level of lncRNA AK12h348, in anoxia/reoxygenation (A/R) primary cardiomyocyte was much higher than that in the Control. LDH release in A/R primary cardiomyocytes was also higher than the Control. After transfection with siRNA-lncRNA AK12348, the release reduced significantly. Reactive oxygen species (ROS) play an important role in the pathogenesis of ischaemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to AR [14]. Lactate dehydrogenase (LDH) has long been considered a useful clinical
marker of intravascular haemolysis [15]. It exists in sera of cancer patients and is taken as a marker for prognosis for patients with pancreatic carcinoma, osteosarcoma, renal or testicular carcinoma [16–18]. The levels of LDH in serum were increased a little in extravascular haemolysis, such as immune haemolytic anaemia, but the extent was high in intravascular haemolysis, such as thrombotic thrombocytopaenic purpura and paroxysmal nocturnal haemoglobinuria [19]. LDH-5 could catalyse the reversible transformation of pyruvate to lactate, which plays a key role in the anaerobic cellular metabolism. In non-small-cell lung cancer, it is associated with tumour hypoxia, angiogenic factor production and poor prognosis [20]. Our study showed AR enhanced LDH release and knockdown of lncRNA AK12348 reduced LDH release. Moreover, the apoptotic rate of cardiomyocytes also decreased after A/R primary cardiomyocyte was transfected with siRNA-lncRNA AK12348. Then, results of western blotting showed AR increased the protein expression levels of PARP and Caspase-3. After transfection with siRNA-lncRNA AK12348, the level decreased. PARP is responsible for post-translational modification of proteins in response to numerous endogenous and environmental genotoxic agents [21]. It is a conserved
Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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nuclear protein that binds rapidly and directly to both singleand double-strand breaks [22]. It constitutes a family of enzymes involved in the regulation of many cellular processes such as DNA repair, gene transcription, cell cycle progression, cell death, chromatin functions and genomic stability [23]. PARP-1 is involved in DNA repair and activated by DNA damage [24]. Inhibition of PARP-1 could increase mitochondrial metabolism through activating SIRT1 [25]. Apoptosis has gained central importance in the study of many biological processes, including neoplasia, neurodegenerative diseases, and development [26]. Caspase-3 has been identified as a key protease in the execution of apoptosis [27]. During programmed cell death, activation of Caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of Caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis [28]. Caspase-3 could activate ROCK-1 which plays an essential role in cardiac myocyte apoptosis [29]. In conclusion, the current results suggest that the upregulation of lncRNA AK12348 expression contributes to myocardial ischaemia-reperfusion injury. Knockdown of lncRNA AK12348 protects myocardium against A/R-induced cardiomyocyte death and apoptosis by targeting PARP. This may provide a promising approach to treating patients suffering from heart disease. However, the present study has limitations. In this study, we only explored the role of lncRNA AK12348 on cardiomyocyte death and apoptosis, the effects of lncRNA AK12348 on cardiomyocyte migration also need be explored, which is important for myocardial ischaemiareperfusion injury repair, cardiomyocyte migrate to injury area and repairing the injury area. Also, the mechanism of lncRNA AK12348 involved in myocardial ischaemia-reperfusion injury in this study was not explored; more markers need to be detected, such as Ki-67, Caspase 6, Bax, Bcl-2, BaxXl. In our future study, we will show these results.
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[18_TD$IF]Acknowledgements
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337 Q28 This study was sponsored by the Program of Education 338 Department[19_TD$IF] of Zhejiang province (grant code: Y201223954
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and Y201534626), Program of Science and Technology[20_TD$IF] of Zhejiang province (grant code: 2015KWB150), And program of Administration of Traditional Chinese Medicine[21_TD$IF] of Zhejiang Province (grant code: 2015ZA047).
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References
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[4] Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nature Reviews Genetics 2009;10(3):155–9. [5] Garzon R, et al. Expression and prognostic impact of lncRNAs in acute myeloid leukemia. Proceedings of the National Academy of Sciences 2014;111(52):18679–84. [6] Guttman M, et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 2011;477(7364):295–300. [7] Hung T, et al. Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters. Nature Genetics 2011;43(7):621–9. [8] Liu Y, et al. Expression profiling and ontology analysis of long noncoding RNAs in post-ischemic heart and their implied roles in ischemia/reperfusion injury. Gene 2014;543(1):15–21. [9] Xie H, et al. Targeting lactate dehydrogenase-A inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells. Cell Metabolism 2014;19(5):795–809. [10] Hamacher-Brady A, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death & Differentiation 2007;14(1):146–57. [11] Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovascular Research 2002;53(1):31–47. [12] De Celle T, et al. Alterations in mouse cardiac proteome after in vivo myocardial infarction: permanent ischaemia versus ischaemia–reperfusion. Experimental Physiology 2005;90(4):593–606. [13] Gross GJ, Auchampach JA. Reperfusion injury: does it exist? Journal of Molecular and Cellular Cardiology 2007;42(1):12–8. [14] Zulueta JJ, et al. Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. American Journal of Physiology-Lung Cellular and Molecular Physiology 1997;272(5): L897–902. [15] Kato GJ, et al. Lactate dehydrogenase as a biomarker of hemolysisassociated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood 2006;107(6):2279–85. [16] Tas F, et al. Prognostic factors in pancreatic carcinoma: serum LDH levels predict survival in metastatic disease. American Journal of Clinical Oncology 2001;24(6):547–50. [17] Ferrari S, et al. Predictive factors of disease-free survival for non-metastatic osteosarcoma of the extremity: an analysis of 300 patients treated at the Rizzoli Institute. Annals of Oncology 2001;12(8):1145–50. [18] von Eyben FE, et al. Serum lactate dehydrogenase isoenzyme 1 and relapse in patients with nonseminomatous testicular germ cell tumors clinical stage I. Acta Oncologica 2001;40(4):536–40. [19] Tabbara IA. Hemolytic anemias. Diagnosis and management. The Medical Clinics of North America 1992;76(3):649–68. [20] Koukourakis M, et al. Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia: angiogenic factor production and poor prognosis. British Journal of Cancer 2003;89(5):877–85. [21] Herceg Z, Wang Z-Q. Functions of poly (ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2001;477(1):97–110. [22] Mun˜oz-Ga´mez JA, et al. PARP inhibition sensitizes p53-deficient breast cancer cells to doxorubicin-induced apoptosis. Biochemical Journal 2005;386(1):119–25. [23] Tentori L, Graziani G. Chemopotentiation by PARP inhibitors in cancer therapy. Pharmacological Research 2005;52(1):25–33. [24] Ying W, Garnier P, Swanson RA. NAD+ repletion prevents PARP-1induced glycolytic blockade and cell death in cultured mouse astrocytes. Biochemical and Biophysical Research Communications 2003;308(4): 809–13. [25] Bai P, et al. PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation. Cell Metabolism 2011;13(4):461–8. [26] Gown AM, Willingham MC. Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. Journal of Histochemistry & Cytochemistry 2002;50 (4):449–54. [27] Blomgren K, et al. Synergistic Activation of Caspase-3 by m-Calpain after Neonatal Hypoxia-Ischemia A MECHANISM OF ‘‘PATHOLOGICAL APOPTOSIS”? Journal of Biological Chemistry 2001;276(13):10191–8. [28] Clark RS, et al. Caspase-3 mediated neuronal death after traumatic brain injury in rats. Journal of Neurochemistry 2000;74(2):740–53. [29] Chang J, et al. Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis. Proceedings of the National Academy of Sciences 2006; 103(39):14495–500.
Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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ORIGINAL ARTICLE
Heart, Lung and Circulation (2017) xx, 1–8 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2017.04.011
Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3
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Chengfei Zheng, Lu Tian, Donglin Li, Ziheng Wu, Xiaohui Wang, Yunjun He, Yangyan He, Wei Jin, Ming Li, Qianqian Zhu, Tao Shang, Hongkun Zhang *
Q2 The First Affiliated Hospital of Zhejiang Medical University, China Received 22 January 2017; received in revised form 11 April 2017; accepted 15 April 2017; online published-ahead-of-print xxx
[1_TD$IF]Backgroud
Recently long non-coding RNAs (lncRNAs) have attracted attention in several biomedical fields. The purpose of this study is to investigate the profile of myocardial lncRNAs and their potential roles in myocardial ischaemia-reperfusion injury (IRI).
Methods
EdgeR bioconductor package was used to screen differentially expressed lncRNAs in myocardial IRI, and lncRNA AK12348 was selected. The mRNA levels of lncRNA AK12348 in normal and anoxia/reoxygenation (A/R) cardiomyocytes were determined by qRT-PCR. After transfection with siRNA-lncRNA, AK12348, LDH release and cell apoptotic rates in normal and A/R cardiomyocytes were determined. The protein expression values of PARP and Caspase-3 were also determined by western blotting.
Q1
Results
The relative level of lncRNA AK12348, LDH release and cell apoptotic rate in A/R cardiomyocytes was significantly higher than that in normal cardiomyocytes. After transfection with siRNA-lncRNA AK12348, LDH release and cell apoptotic rates in A/R cardiomyocytes were reduced, while the values in normal cardiomyocytes had almost no change. The protein expression values of PARP and Caspase-3 in A/R cardiomyocytes were much higher than the Control. After knockdown of lncRNA AK12348, the values decreased.
Q1
Conclusion
Long non-coding RNAs AK12348 could be potential therapeutic targets for the treatment of myocardial IRI.
Keywords
LncRNA AK12348 Myocardial IRI A/R LDH PARP
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Introduction Q12
Coronary heart disease (CHD) is the leading cause of death and disability in the world. According to the WHO, 7,254,000 deaths worldwide (12.8% of all deaths) resulted from CHD in 2008. The effects of CHD are usually caused by acute myocardial ischaemia-reperfusion injury (IRI) [1]. Ischaemiareperfusion injury is a complex phenomenon involving many
players, all contributing to the final damage inflicted on the heart [2]. Ischaemia-reperfusion causes damage to membrane phospholipids, and is a significant mechanism of cardiac I/R injury. Molecular dissection of sarcolemmal damage in I/R, however, has been difficult to address experimentally [3]. Long non-coding RNAs are RNAs that are longer than 200 nucleotides in length and do not code for proteins, although
*Corresponding author. Email: [email protected] © 2017 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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they can interact with proteins [4]. They are involved in numerous biological roles including epigenetic regulation, apoptosis, and cell cycle [5]. Emerging evidence suggests that lncRNAs may serve as master gene regulators capable of controlling protein-coding and non-coding genes and, as such, they have been implicated in the regulation of a variety of cellular functions and disease processes including stemness and cancer metastasis [6,7]. A study by Liu et al. showed gene ontology and pathway analysis indicated lncRNAs play important roles in the post-ischaemic heart [8]. However, further exploration of the effect of lncRNAs on myocardial IRI has not been conducted. In this study, we screened the differentially expressed lncRNAs in primary cardiomyocytes with myocardial ischaemia-reperfusion injury. The effect of lncRNA on the proliferation of cardiomyocytes was studied and the molecular mechanism was explored.
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Material and Methods
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Identification of Differentially Expressed lncRNA
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The microarray expression profiles of GSE50378 and GSE22282 were downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database. Gene expression profiles between them were compared. We transferred the probe-level data in CEL files into expression measures. Then, background was corrected and quartile data was normalised by the robust multiarray average (RMA) algorithm. The file in the platform annotation files provided by Affymetrix Company was used to map the relationship between the probes and gene symbols. A probe would be filtered if the probe did not have the corresponding gene symbol. The average value of the gene symbol with multiple probes obtained would be further analysed. The primary comparison of samples from HS patients and healthy donors was performed by Linear Models for Microarray Data (LIMMA) package. A set of gene-specific t tests with the threshold of false discovery rate (FDR) 0.05 was assimilated to identify differentially expressed lncRNAs.
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Tissue Samples and Cell Culture
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Fifty-five pairs of tissues were collected from rats with myocardial ischaemia/reperfusion injury and normal rats. After isolation, the tissues were stored at 80 C. Rat cardiomyocyte H9C2 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were kept in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) in humidified air containing 5% CO2 at 37 C. Dulbecco’s Modified Eagle’s Medium contained 10% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin/ streptomycin.
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Cell Transfection
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RNA interference (RNAi) is an evolutionarily conserved surveillance mechanism that responds to double-stranded
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Q18
RNA by sequence-specific silencing of homologous genes. To investigate the role of lncRNA AK12348 on myocardial cells, we used a RNAi-based strategy (c-lncRNA AK12348-siRNA) to specifically silence lncRNA AK12348 expression. LncRNA AK12348-siRNA transfection (50 nmol/L) was performed in cultured myocardial cell H9C2. Then, qRT-PCR was used to detect relative mRNA level.
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RNA Isolation and Quantitative Realtime Reverse Transcription-PCR (qRTPCR)
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TRIzol reagent[12_TD$IF] (Invitrogen) was used to isolate total RNA from tissues or cells. Total RNA was used to synthesise cDNA with PrimeScript reverse transcription-PCR kit (TaKaRa, Shiga, Japan), and subjected to quantitative realtime PCR. The relative expression levels of miR-181a were measured using a SYBR PrimeScript miRNA quantitative real-time polymerase chain reaction Kit (TaKaRa, Shiga, Japan) following the manufacturer’s instructions, with U6 as an internal control. MiR-181a mRNA expression levels were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), with GADPH as an internal control. The reaction was performed on an ABI 7500 Sequence Detection System (ABI, Vernon, CA, USA).
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Western Blotting
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Total proteins from the cells were extracted by ice-cold RIPA lysis buffer supplemented with 1 mM proteinase inhibitor PMSF (Sigma, St. Louis, MO, USA). The protein concentration was quantified with a BCA assay kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF, Millipore, Bedford, MA, USA) membrane, and then blocked with 5% non-fat milk in Tris-buffered saline. The membranes were incubated with primary antibodies, mouse antihuman monoclonal ZEB1, E-cadherin, Vimentin and N-cadherin antibody (Santa Cruz Biotechnology, CA, USA) and mouse anti-human monoclonal b-acitin antibody (Santa Cruz Biotechnology, CA, USA), at 4 C overnight. The membranes were washed and subsequently probed with secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1/4000 dilution for 1 h at room temperature. Proteins were visualised with chemiluminescent detection system (ECL; Beyotime). GAPDH was used as internal control.
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Flow Cytometry Assay
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Flow cytometry assay was used to clarify cell apoptosis. Cells were collected with trypsinisation and then washed twice with PBS, and fixed in cold 80% ethanol, and finally stored at 4 C overnight. The cells were washed with PBS and RNase A was administered. Propidium iodide was added to tubes and then incubated for 20 min at 4 C in the dark. FITC-labelled Annexin V/PI staining was applied. 1 106 cells in each well were suspended with buffer containing FITC-conjugated
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Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Annexin V/PI. Samples were then analysed via flow cytometry.
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Immunohistochemistry
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Cell histologic sections were deparaffinised, rehydrated, and blocked with methanolic 3% hydrogen peroxide. The slides were immersed in 10 mm citrate buffer, pH 6.0 and heated in a 1200 W microwave oven at the highest power setting. Evaporated liquid was replenished at 5 mins, then the slides were heated at high power for an additional 5 mins. The slides were left in the buffer for an additional 10 mins before being removed. The antibody used was a mouse monoclonal antibody raised against full-length recombinant Cdx2 protein (AM392–5 m; BioGenex, San Ramon, CA). Immunostaining was performed by hand using the prediluted antibody solution and a 30-min incubation. After washing, antibody binding was visualised using the avidin-biotinperoxidase technique (Vectastain Elite ABC kit, Vector Laboratories, Burlington, VT), followed by incubation with [13_TD$IF]3,30 diaminobenzidine tetrahydrochloride. The slides were counterstained with haematoxylin.
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Statistical Analysis
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Statistical analyses were performed using SPSS Statistics (SPSS Inc., Chicago, IL, USA). All data were expressed as
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mean standard deviation. Student[14_TD$IF]’s t-test or one-way ANOVA test was performed to determine significant differences. A p ＜ 0.05 was considered statistically significant.
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Results
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Screening of Differentially Expressed lncRNA
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To explore the lncRNAs that play key roles in myocardial IRI, lncRNAs data were downloaded from the database and uploaded to GEO (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi[25_TD$IF]? acc=GSE50378; http://www.ncb i.nlm.nih. gov/geo/query/acc.cgi?acc[16_TD$IF]=GSE22282) to screen differentially expressed genes. Results showed 26 differentially expressed lncRNAs were taken as representative in GSE50378, of them, eight lncRNAs were up-regulated by more than 1.5-fold, and 18 miRNAs were down-regulated by more than 1.5-fold. In GSE22282, 65 differentially expressed lncRNAs were taken as representative. Of those, 30 were down-regulated and 35 were up-regulated ([1_TD$IF]Figure 1). The top 26 differentially expressed lncRNAs in GSE50378 are shown in Table 1a, the differentially expressed lncRNAs in GSE22282 are shown in Table 1b. There are 164 differentially expressed lncRNAs in GSE50378 and 119 differentially
[1_TD$IF]Figure 1 Heatmap of lncRNAs in myocardial ischaemia-reperfusion injury tissues that was significantly down-regulated or up-regulated. For each miRNA the red colour means an up-regulated expression, and the blue colour means a downregulated expression. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Table 1a Top 26 differentially expressed lncRNAs in GSE50378 samples. GSE50378
p Value
AK123483 AK024898
3.779478 2.202297
1.67E-08 1.43E-06
AL389942
1.743006
4.30E-06
BC004287
1.552815
6.80E-06
AK093009
1.744483
1.71E-05
AL110176
1.626017
2.26E-05
AK123079
1.341703
4.78E-05
AK091312
1.105875
6.17E-05
CR611122 BC013423
1.31421 1.095659
6.32E-05 7.52E-05
AK123657
1.00934
7.56E-05
AK023774 CR613326
180
logFC
1.341952 1.13839
9.38E-05 9.82E-05
BC020900
1.050885
0.000108
AK021543
1.107317
0.000115
BC043185
1.047101
0.000126
AY605064 AK023526
1.10054 1.768026
0.000143 0.00022
AK055670
1.0225
0.000309
EU249757
1.02182
0.000324
AK058186
1.04111
0.000354
AJ227892
1.432
0.000376
BC041955
1.050115
0.000451
AK057923
1.292861
0.002547
BC043182 BC018139
1.286492 1.20351
0.007411 0.008528
182
expressed lncRNAs in GSE22282. Among them, four lncRNAs were the same and they were AF495725, AK098081, AK025695 and BC023972 (Table 2).
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Myocardial Cell Identification
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To explore the effect of lncRNA on rat cardiomyocytes more precisely, we isolated cardiomyocytes to culture for the further study. Immunofluorescence assay was used to determine the purity of cultured primary cardiomyocytes. The immunofluorescence result of cardiac troponin T (cTnT) showed the cytoplasm of cardiomyocytes presented green fluorescence, which indicated immunofluorescence staining was positive ([1_TD$IF]Figure 2A). It indicated the purity of cultured primary cardiomyocytes was well and could be used for the next experiment. The mRNA levels of lncRNA AK123483 in normal primary cardiomyocytes (Control) and cells with anoxia/reoxygenation were determined by qRT-PCR. Results showed the level of lncRNA AK123483 in the A/R group was significantly higher than that in the Control ([1_TD$IF] Figure 2b).
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Cell Transfection
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We used the lentivirus pGFP-V-RS-lncRNA AK123483 siRNA and pGFP-V-RS vector to infect cardiomyocyte
201
H9C2. The transfection efficiency was also determined by fluorescence microscope, and results showed most cells were transfected with pGFP-V-RS-lncRNA AK123483 siRNA in the silncRNA AK123483 groups ([1_TD$IF]Figure 3a). The mRNA levels of lncRNA AK123483 in the Control, si-Con, siRNA1, siRNA2 and siRNA3 groups were determined by qRT-PCR. Results showed the mRNA levels of lncRNA AK123483 in the siRNA1, siRNA2 and siRNA3 groups were significantly higher than that in the Con and siCon groups ([1_TD$IF]Figure 3b).
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Knockdown of lncRNA AK123483 Influenced LDH Release and Cell Apoptosis
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The lactate dehydrogenase-A (LDH-A) enzyme catalyses the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumour outcomes [9]. In this study, results showed knockdown of lncRNA AK123483 had no influence on LDH release in primary cardiomyocyte (Control); LDH release in A/R primary cardiomyocyte was much higher than that in the Control. After transfection with siRNA-lncRNA AK123483, LDH release decreased significantly ([1_TD$IF]Figure 4a). The influence of lncRNA AK123483 expression on the apoptosis of cardiomyocytes at A/R condition was also determined by flow cytometry. Results showed knockdown of lncRNA AK123483 had no influence on apoptotic rate in the Control group. The apoptotic rate in the A/R group was markedly higher than that in the Control. After A/R primary cardiomyocyte was transfected with siRNA-lncRNA AK123483, the apoptotic rate decreased dramatically ([1_TD$IF]Figure 4b and c).
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212 213 215 216 217 218 219 220 221 222 223 Q22
224 225 226 227 228 229 230
Knockdown of lncRNA AK123483 Regulated the Expression of PARP
231
As the above results show, A/R could promote the apoptosis of primary cardiomyocytes, and knockdown of lncRNA AK123483 reduced the apoptotic rate. However, the molecular mechanism was unknown. The protein expression values of PARP and Caspase-3 in normal primary cardiomyocyte, and A/R primary cardiomyocyte before and after transfection with siRNA were determined by western blotting. Results show the protein expression values of PARP and Caspase-3 in the A/R group were much higher than the Control. After A/R primary cardiomyocyte was transfected with siRNA, the values decreased ([17_TD$IF]Figure 4b).
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Discussion
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Ischaemic heart disease is a major cause of death and often caused by coronary artery disease. Comprehensively deleted cardiomyocytes is related to ischaemia and reperfusion injury (IRI) [10]. Myocardial IRI could result in many inflammatory responses including production of oxidants, activation of complement, and infiltration by polymorphonuclear neutrophils [11]. Cardiomyocyte death/apoptosis plays an important role in ischaemic hearts. As reported, various genes are abnormally expressed in infarcted hearts, which
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Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Regulation of Myocardial Ischaemia-Reperfusion Injury
Table 1b Differentially expressed lncRNAs in GSE22282 samples. GSE22282
logFC
p Value
logFC
p Value
logFC
p Value
0.834103 1.43372
0.019611 0.019696
0.774723
0.020929
BC053563 BC024020
1.336855 1.638161
4.07E-05 4.64E-05
BC031342 AF318321
1.2318 1.100853
0.011321 0.011347
AK126861 AK098081
U94902
1.406273
9.22E-05
AF086441
1.363236
0.011508
AK291479
AY216265
2.255815
0.000743
AF052160
0.76307
0.011545
AK026440
0.43023
0.021097
0.001072
BC000986
0.50081
0.011714
BC037530
0.52414
0.021661
AK094088
3.80262
AF495725
1.116279
0.001094
AK002146
2.08507
0.011897
AK090808
0.76373
0.023217
BC014583
0.913022
0.001272
AB007954
0.63759
0.0125
AK054868
0.60838
0.023446
BC040834
2.073123
0.002196
AK024965
0.012593
BC039682
1.45356
0.024294
AF086543 AK057657
1.148865 0.64271
0.003023 0.003606
AF075045 AK054971
1.32901 0.66463
0.012735 0.013026
U72512 AK095104
0.54101 1.383514
0.024664 0.025161
BC006384
0.784642
0.003789
AX721088
0.56321
BC018655
0.831002
0.004952
AK021459
AK092600 AK025695
0.57829
0.005344
AF090921
0.826167
0.005639
AK025166
0.971376
0.813113
0.01349
AL049450
0.4768
0.025646
0.843602
0.013912
BC041399
0.3878
0.026269
0.978045
1.36545
0.013928
AK002088
0.62692
0.014248
CR595807
0.0144
0.006468
AK022196
1.31242
AK311445
0.57075
0.006541
BC035078
0.47204
AK024385 AK026372
2.45816 0.935024
0.006929 0.007245
AF147344 AK090404
BC039325
1.28851
U94903
AK021555 AK021643 BC037234
0.467489 1.29959 1.008319
0.02638 0.026814
0.015331
0.740554 0.70235
0.015621 0.015742
0.008109
uc003lfc.1
0.54317
0.015788
0.009064
uc004ebu.1
0.54809
0.0159
0.009288
AL109779
1.14044
0.017035
0.009724
AK021803
1.40662
0.017181
AK023330
1.373985
0.009752
AK123379
0.6377
0.017608
AB019562
2.045813
0.009763
AK022157
0.53792
0.018188
AK130460 AK091637
0.59566 0.960152
0.010953 0.010955
AK123861
0.811307
1.687597
0.018856
Figure 2 A. Immunofluorescence results of cultured primary cardiomyocyte. B. Expression level of lncRNA AK12348 in normal and A/R primary cardiomyocyte. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Table 2 Differentially expressed lncRNAs in both GSE50378 and GSE22282. logFC
GSE50378
GSE22282
p Value
Average expression
Average expression
level (normoxial)
level (hypoxial)
AF495725
0.529028
0.003148
8.53034
9.059368
[5_TD$IF]AK098081
0.64122
0.003381
9.751949
9.110731
[6_TD$IF]AK025695
0.72119
0.001259
9.699084
8.977898
[7_TD$IF]BC023972
0.78286
0.00049
4.09202
3.309155
AF495725
1.116279
0.001094
6.911135
8.027415
[8_TD$IF]AK098081
1.43372
0.019696
7.972042
6.538321
[9_TD$IF]AK025695
0.826167
0.005639
8.881449
9.707616
[10_TD$IF]BC023972
0.597165
0.030373
5.020953
5.618119
Figure 3 Knockdown of lncRNA AK12348 influenced LDH release. A. Normal and A/R primary cardiomyocyte were transfected with siRNA-lncRNA AK12348. The transfection efficiency was determined by fluorescence microscope. B. LDH release in normal and A/R primary cardiomyocyte before and after transfection with siRNA- lncRNA AK12348. Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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Figure 4 Knockdown of lncRNA AK12348 influenced the apoptosis primary cardiomyocytes. A. The apoptosis situation of normal and A/R primary cardiomyocytes before and after transfection with siRNA-lncRNA AK12348. B. The protein expression values of PARP and Caspase-3 in normal and A/R primary cardiomyocytes before and after transfection with siRNA-lncRNA AK12348.
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can result in cardiac remodelling after IRI [12]. The cellular mechanisms underlying IRI are complex and involve many signalling pathways and molecular players [13]. Therefore, it is necessary to find multiple molecular targets. Since lncRNAs are found to regulate gene expression, it is reasonable to suppose that lncRNAs may play an important role in IRI. In this study, we screened the differentially expressed lncRNAs in GSE50378 and GSE22282, which were downloaded from GEO, and the top 26 differentially expressed genes were obtained. LncRNA AK12348 was the most differentially expressed gene in GSE50378 and selected for the further study. We obtained the relative level of lncRNA AK12h348, in anoxia/reoxygenation (A/R) primary cardiomyocyte was much higher than that in the Control. LDH release in A/R primary cardiomyocytes was also higher than the Control. After transfection with siRNA-lncRNA AK12348, the release reduced significantly. Reactive oxygen species (ROS) play an important role in the pathogenesis of ischaemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to AR [14]. Lactate dehydrogenase (LDH) has long been considered a useful clinical
marker of intravascular haemolysis [15]. It exists in sera of cancer patients and is taken as a marker for prognosis for patients with pancreatic carcinoma, osteosarcoma, renal or testicular carcinoma [16–18]. The levels of LDH in serum were increased a little in extravascular haemolysis, such as immune haemolytic anaemia, but the extent was high in intravascular haemolysis, such as thrombotic thrombocytopaenic purpura and paroxysmal nocturnal haemoglobinuria [19]. LDH-5 could catalyse the reversible transformation of pyruvate to lactate, which plays a key role in the anaerobic cellular metabolism. In non-small-cell lung cancer, it is associated with tumour hypoxia, angiogenic factor production and poor prognosis [20]. Our study showed AR enhanced LDH release and knockdown of lncRNA AK12348 reduced LDH release. Moreover, the apoptotic rate of cardiomyocytes also decreased after A/R primary cardiomyocyte was transfected with siRNA-lncRNA AK12348. Then, results of western blotting showed AR increased the protein expression levels of PARP and Caspase-3. After transfection with siRNA-lncRNA AK12348, the level decreased. PARP is responsible for post-translational modification of proteins in response to numerous endogenous and environmental genotoxic agents [21]. It is a conserved
Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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nuclear protein that binds rapidly and directly to both singleand double-strand breaks [22]. It constitutes a family of enzymes involved in the regulation of many cellular processes such as DNA repair, gene transcription, cell cycle progression, cell death, chromatin functions and genomic stability [23]. PARP-1 is involved in DNA repair and activated by DNA damage [24]. Inhibition of PARP-1 could increase mitochondrial metabolism through activating SIRT1 [25]. Apoptosis has gained central importance in the study of many biological processes, including neoplasia, neurodegenerative diseases, and development [26]. Caspase-3 has been identified as a key protease in the execution of apoptosis [27]. During programmed cell death, activation of Caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of Caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis [28]. Caspase-3 could activate ROCK-1 which plays an essential role in cardiac myocyte apoptosis [29]. In conclusion, the current results suggest that the upregulation of lncRNA AK12348 expression contributes to myocardial ischaemia-reperfusion injury. Knockdown of lncRNA AK12348 protects myocardium against A/R-induced cardiomyocyte death and apoptosis by targeting PARP. This may provide a promising approach to treating patients suffering from heart disease. However, the present study has limitations. In this study, we only explored the role of lncRNA AK12348 on cardiomyocyte death and apoptosis, the effects of lncRNA AK12348 on cardiomyocyte migration also need be explored, which is important for myocardial ischaemiareperfusion injury repair, cardiomyocyte migrate to injury area and repairing the injury area. Also, the mechanism of lncRNA AK12348 involved in myocardial ischaemia-reperfusion injury in this study was not explored; more markers need to be detected, such as Ki-67, Caspase 6, Bax, Bcl-2, BaxXl. In our future study, we will show these results.
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[18_TD$IF]Acknowledgements
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337 Q28 This study was sponsored by the Program of Education 338 Department[19_TD$IF] of Zhejiang province (grant code: Y201223954
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and Y201534626), Program of Science and Technology[20_TD$IF] of Zhejiang province (grant code: 2015KWB150), And program of Administration of Traditional Chinese Medicine[21_TD$IF] of Zhejiang Province (grant code: 2015ZA047).
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Please cite this article in press as: Zheng C, et al. Long Noncoding RNA AK12348 is Involved in the Regulation of Myocardial Ischaemia-Reperfusion Injury by Targeting PARP and Caspase-3. Heart, Lung and Circulation (2017), http://dx.doi.org/ 10.1016/j.hlc.2017.04.011
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