p53 axis

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LncRNA UCA1 protects cardiomyocytes against hypoxia/reoxygenation induced apoptosis through inhibiting miR-143/MDM2/p53 axis Qiang-Sheng Wanga, Jun Zhoub, Xun Lia, a b

⁎

Department of Vasculocardiology, The First Affiliated Hospital of Soochow University, Soochow 215006, PR China Department of Vasculocardiology, The Affiliated Tongren Hospital of Shanghai Jiaotong University of Medical College, Shanghai 200336, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Hypoxia/reoxygenation lncUCA1 miR-143 MDM2 p53 Apoptosis

Background: lncUCA1 is abundantly expressed in the heart, indicating it may be important in maintaining normal myocardial function. However, the underlying mechanism of lncUCA1 in heart disease, particularly myocardial infarction (MI), is still in its infancy. Methods: LncUCA1 and miR-143 expression were measured in hearts of MI models. Overexpression and knockdown of lncUCA1 in neonatal rat cardiomyocytes were performed to confirm the effects of lncUCA1 in hypoxia-induced apoptosis. Results: The expression of lncUCA1 decreased but miR-143 increased inversely in MI heart. Overexpressing lncUCA1 protected cardiomyocytes from H/R induced apoptosis via inhibiting miR-143, which regulates apoptosis by targeting MDM2/p53 pathway. While silencing lncUCA1 caused miR-143 upregulation and H/Rinduced apoptosis increase. Moreover, miR-143 was proved to be a competitive target of lncUCA1. Conclusions: lncUCA1 might protect cardiomyocyte against H/R induced apoptosis by suppressing miR-143 and modulated the following downstream MDM2/p53 signaling pathway, indicating the therapeutic potential of targeting lncUCA1 for MI.

1. Introduction Ischemic heart disease is the leading cause of death in the world. Acute myocardial infarction is a fatal ischemic heart disease, and reperfusion therapy is an effective treatment for it. However, many patients suffer from irreversible myocardial ischemia/reperfusion injury (MIRI) after reperfusion therapy. Therefore, novel therapies are required to prevent and treat MIRI. Non-coding RNA is a class of regulatory RNA that does not encode protein but participate in regulating other genes expression. A large number of studies have confirmed that non-coding RNA expression abnormally in most of human diseases, such as neurodegenerative diseases and cancer [1,2], also including cardiovascular disease [3,4]. LncRNAs, > 200 nucleotides in length, participate in many biological and pathological processes [5,6], and deep sequencing revealed that the profile of myocardial lncRNAs is altered upon heart failure in humans [7]. The first lncRNA identified as a risk marker is the MIAT1 (myocardial infarction associated transcript 1), which remarkably upregulated in acute myocardial infarction (AMI) patients and animal models [8,9], promoting cardiomyocytes apoptosis and inflammatory cell

infiltration [10], involving in regulation of cardiac fibrosis [11]. Other more lncRNAs were found successively, such as H19, ARF, NRF and UCA1 and so on [4,5]. Urothelial carcinoma-associated 1 (UCA1), an lncRNA originally found over-expressed in bladder transitional cell carcinoma, is a wellknown cancer-promoting gene [12,13]. Subsequently, researchers detected that lncUCA1 is expressed specifically and abundantly in heart of adult, indicating that lncUCA1 may play important roles in maintaining normal myocardial function and may be as a biomarker for heart diseases, AMI for example [12,14]. Recent research showed that the plasma lncUCA1 level is decreased at the early state of AMI patients and increased at day 3 after AMI, inversely associating with the miR-1 expression [14]. Another study demonstrated the reduced expression of lncUCA1 in MIRI rat models, promoting H2O2-induced cardiomyocyte apoptosis partially through stimulation of p27 protein expression [15]. While hypoxia/reoxygenation (H/R)–induced cardiomyocyte apoptosis is one of the major causes of MIRI, suggesting that lncUCA1 may be as a predictive biomarker for the diagnosis and prognosis of MI. However, the further regulation mechanism of lncUCA1 in MIRI is still a mystery. In this study, we illuminated a novel regulatory mechanism of

⁎ Corresponding author at: Department of Vasculocardiology, The First Affiliated Hospital of Soochow University, No. 188, Shizi Street, Soochow 215006, Jiangsu Province, PR China. E-mail address: [email protected] (X. Li).

https://doi.org/10.1016/j.ygeno.2019.04.009 Received 11 September 2018; Received in revised form 8 April 2019; Accepted 13 April 2019 0888-7543/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Qiang-Sheng Wang, Jun Zhou and Xun Li, Genomics, https://doi.org/10.1016/j.ygeno.2019.04.009

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air and 5% CO2).

lncUCA1 in the pathological process of MIRI. We found that an inverse expression profile between lncUCA1 and miR-143 in both MIRI rat models and neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R). Moreover, we demonstrated that miR-143 was a competitive inhibiting target of lncUCA1 by direct binding with each other, and lncUCA1 might protected cardiomyocytes against H/R-induced cardiomyocyte apoptosis by targeting inhibition of miR-143/ MDM2/p53 axis. Our findings identified a novel mechanism involving lncUCA1, miR-143 and MDM2/p53 in regulating H/R-induced cardiomyocyte apoptosis. Modulation of their levels may provide novel therapeutic strategies for myocardial infarction and heart failure.

2.4. MiRNA oligonucleotides, lentivirus preparation and cell transfection A Lenti-X™ Lentiviral Expression System (Clontech, Japan) was used to prepare lncUCA1-overexpressed and shRNA lentivirus according to the manufacturer's introductions. Briefly, Gene transfer into the lenti-X vectors. The UCA1 gene (1.4 kb isoform) was cloned into an over-expression vector pLVX-Puro containing a cytomegalovirus (CMV) promoter. Three short hairpin RNAs (shRNAs) were designed by online software BLOCK-iT™ RNAi Designer (Invitrogen, USA) and subcloned into a vector pLVX-shRNA1 containing a U6 promoter. The control virus without UCA1 cDNA or shRNA was prepared separately. Then, the recombinant Lenti-X vectors were co-transfected respectively with Lenti-X Packaging Single Shots into Lenti-X 293 T Packaging cells to generate lentivirus according the guidelines. Small RNA oligonucleotides the mimics and inhibitor of miR-143, with their corresponding negative controls, were designed and synthesized by GenePharma (Shanghai, China), and transfected into cardiomyocytes by Lipofectamine 3000 (Invitrogen, USA) according to the instructions.

2. Materials and methods 2.1. MIRI rat model by coronary artery ligation Sprague-Dawley rats (210–230 g) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). They were housed in the animal room (temperature 25 °C ± 1 °C, humidity 65% ± 5%, and 12-h light/dark cycle). The animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the ethics committee of The First Affiliated Hospital of Soochow University. The myocardial ischemia rat models were prepared by surgery of coronary artery ligation as previously described [16]. Briefly, rats were anesthetized by sodium pentobarbital (90 mg/kg) and their chests were opened to expose the heart. The left anterior descending coronary artery (LAD) was ligated with an 8–0 nylon suture at 2 to 3 mm below the border between left auricle and ventricle to create MI. After 45 min ischemia, the vessel was reopened and reperfused for 3 h or 1 week. The control group performed sham operation with left thoracotomy only. The heart samples were rapidly excised and sectioned into several segments for further experiments.

Flow cytometry was performed to determine cell apoptosis using a Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, USA) according to manufacturer instruction. After treatment, the cells were harvested and washed twice by cold PBS. 1 × 106 cells were suspended in 200 μl binding buffer including 10 μl Annexin V-FITC and 5 μl propidium iodide, followed by incubation in the dark for 30 min. Finally, the cells were measured within 1 h using a flow cytometry (Beckman Coulter, USA), and the data were analyzed by the FlowJo software (LLC, USA).

2.2. Histological analysis

2.6. Total RNA isolation, reverse transcription, and real-time PCR

To evaluate the effectiveness for MIRI model in this study, the heart infarct size was determinated by 2,3,5-triphenyltetrazolium chloride (TTC) staining as previously described [17]. Briefly, the excisional heart samples of rats were rapidly frozen for 20 min, and then cut along serially cross section into about 2-mm slices and stained with 1.0% TTC (Sigma) for 10 min at 37 °C. The size of infarct region was assessed by a blinded observer using computer-assisted planimetry (NIH Image 1.57). A TUNEL assay was performed by using a DeadEnd Fluorometric TUNEL kit (Promega, USA) on paraffin cardiac sections according to the manufacturer's instruction and TUNEL-positive nuclei per section were counted by Image-Pro Plus software.

Total RNA was extracted by TRIzol reagent (Invitrogen, USA) and then was reverse-transcribed into cDNA using a first strand cDNA kit (Sigma, USA). Quantitative real-time PCR was performed with the SYBR Premix Ex Taq™ kit (Takara Bio, China) and run on a 7500 Thermocycler (Applied Biosystems, USA). The relative expression levels of genes were calculated through a 2-ΔΔCt method normalized by GAPDH.

2.5. Flow cytometry analysis

2.7. Immunoblotting analysis Primary antibodies used in this study were purchased from Abcam (England), except for the β-actin antibody (CST, USA). Total proteins were extracted using a RIPA Lysis Buffer (Thermo Scientific, USA). Equal amount of protein was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to a PVDF membrane (Millipore, USA). After blocking with 5% milk for 1 h at room temperature, the membranes were incubated overnight with specific primary antibody (1:1000) at 4 °C. Immunodetection of protein bands was achieved using an ECL Plus detection system (Millipore, USA) according to the instruction. The relative expression was calculated by integrated gray values of the bands normalized with the internal reference protein β-actin.

2.3. Cardiomyocyte isolation, culture and hypoxia/reoxygenation (H/R) treatment Cardiomyocytes were isolated from 1- to 2-days-old rats. Briefly, after dissection hearts were washed and minced in HEPES buffer saline solution. Tissues were dispersed by incubation at 37 °C in HEPES buffer containing 1.2 mg/ml pancreatin (Sigma, USA) and 0.14 mg/ml collagenase II (Gibco, USA). After filtration and centrifugation, the cell precipitates were resuspended in DMEM/F12 medium (Gibco, USA) containing 5% heat-inactivated fatal calf serum, 0.1 mM ascorbate, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.1 mM bromodeoxyuridine, followed by further purification using a differential adhesion method at 37 °C for 1 h. Finally, the unadherent cardiomyocytes were transferred to other new plates with an optimal density for culture. To simulate MIRI in vitro, cardiomyocytes were challenged with a hypoxia/reoxygenation (H/R) treatment in a tri-gas incubator for 6 h hypoxia induction (95% N2 and 5% CO2) and 12 h reoxygenation (95%

2.8. Vector construction and dual luciferase reporter assay The UCA1 gene sequence containing predicted interaction sites, along with the corresponding mutant sites prepared by a QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA), were cloned into the downstream of the Firefly luciferase gene of pmiRreport vector (Ambion, USA). 2

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When HEK-293T cells were cultured to 80% confluence, the recombinant reporter constructs were transfected respectively by lipofectamine 3000, along with miRNA mimics, inhibitors or negative controls. A plasmid pRL-TK carrying the Renilla luciferase gene was also transfected to every group as internal reference of transfection efficiency. After transfection of 24 h, the luciferase activities were measured using a Dual-Luciferase Reporter Assay kit (Promega, USA) following the manufacturer's instructions. The relative luciferase activity of every treatment was calculated by the rate of firefly luciferase activity to Renilla luciferase activity.

more, the expression of lncUCA1 and miR-134 were observed in an inverse changing trend, with significant downregulation of lncUCA1 but upregulation of miR-143, in the MIRI hearts compared with Sham group (Fig. 1C). Additionally, the expression of anti-apoptotic protein Bcl-2 was decreased, while pro-apoptotic proteins Bax and Caspase-3 were increased (Fig. 1 D). These results indicated that downregulated lncUCA1 might play a role in cardiomyocyte apoptosis of MIRI rats.

2.9. Statistical analysis

To explore the effects of lncUCA1 on H/R induced cardiomyocyte apoptosis, recombinant lentivirus carrying lncUCA1 cDNA (LvlncUCA1) or shRNAs (Lv-shlncUCA1) were used for overexpression or knockdown in vitro. Q-PCR was performed to verify the efficiency of these recombinant lentivirus, coming out the most effective LvshlncUCA1-2 selected for the following study (Fig. 2A). H/R treatment significantly decreased the expression of lncUCA1 and induced cardiomyocyte apoptosis (Fig. 2B and C). LncUCA1 overexpressed-lentivirus (Lv-lncUCA1) observably inhibited or slowed down the effects of H/R treatment, while lncUCA1 shRNA-lentivirus (Lv-shlncUCA1) significantly promoted these phenomena (Fig. 2B and C). The expression of apoptosis related proteins also changed correspondingly (Fig. 2D). These results indicated that lncUCA1 inhibited H/R induced cardiomyocyte apoptosis.

3.2. LncUCA1 involved in the regulation of cardiomyocyte apoptosis induced by H/R

Each experiment was performed at least three times with consistent results, and data were presented as mean ± standard deviation (SD). The Student's t-test or one-way ANOVA was used for differences analysis and p-value < .05 was considered as a statistically significant difference. 3. Results 3.1. The downregulation of lncUCA1 and upregulation of miR-143 related to cardiomyocyte apoptosis in MIRI rat hearts To determine the potential role of lncUCA1 in MIRI injury, we established a MIRI rat model by surgery of coronary artery ligation. The rats' heart samples were collected to determinate myocardial infarction area by TTC staining and apoptosis ratio by TUNEL assay for evaluating the effectiveness of MIRI. These results showed that remarkably increased infarcted area and cardiomyocyte apoptosis occurred in the MIRI heart samples compared with controls (Fig. 1A and B). What's

3.3. MiR-143 contributed to H/R-induced cardiomyocyte apoptosis by suppressing MDM2 and activating p53 signal pathway Since miR-143 was upregulated in MIRI rat heart identified above, the function mechanism of miR-143 in H/R-induced cardiomyocyte

Fig. 1. The downregulation of lncUCA1 and upregulation of miR-143 related to cardiomyocyte apoptosis in MIRI rat hearts. (A) The myocardial infarct size of MIRI rats by coronary artery ligation and sham group with operation of left thoracotomy only, by TTC staining. (B) Representative pictures of cardiomyocyte apoptosis ratio were measured by TUNEL staining and the statistical analysis of apoptotic positive rate was displayed in the histogram. (C) qRT-PCR analysis of the expression of lncUCA1 and miR-143 in heart samples from Sham and MIRI rats. (D) Immunoblotting analysis of apoptosis marker proteins Bcl-2, Bax and Caspase-3 expression in Sham and MIRI rat heart samples. The asterisks show difference significant with the Sham group: ** p < .01. 3

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Fig. 2. LncUCA1 involved in the regulation of cardiomyocyte apoptosis induced by H/R. (A) qRT-PCR analysis of the expression of lncUCA1 after recombinant lentivirus treatment for the efficiency verification of lncUCA1 overexpression (left) and knockdown (right) in cardiomyocytes. (B) qRT-PCR analysis of the expression of lncUCA1 in cardiomyocytes co-treated with recombinant lentivirus and H/R challenge. (C) Cell apoptosis analysis by Annexin-V/PI and flow cytometry in cardiomyocytes co-treated with lncUCA1-recombinant lentivirus and H/R challenge. Statistical analysis of the apoptotic positive rate was displayed in the histogram. (D) Immunoblotting analysis of apoptosis marker protein Bcl-2, Bax and Caspase-3 expression in cardiomyocytes co-treated with recombinant lentivirus and H/R challenge. The asterisks show difference significant as *p < .05, **p < .01, ***p < .001.

Fig. 3. MiR-143 promoted H/R induced cardiomyocyte apoptosis through targeting MDM2/p53/Bcl-2 signal pathway. (A) Immunoblotting analysis of MDM2, t-p53 and phosph-p53 in cardiomyocytes transfected with miR-143 mimics (miR-143) or inhibitor (anti-miR-143). The quantitative analysis of protein bands gray intensity was displayed in the histogram (right). (B) Cell apoptosis analysis by Annexin-V/PI and flow cytometry in cardiomyocytes co-treated with miR-143 mimics/inhibitor and H/R challenge. Statistical analysis of the apoptotic positive rate was displayed in the histogram. (C) Immunoblotting analysis of apoptosis marker proteins Bcl-2, Bax and Caspase-3 expression in cardiomyocytes co-treated with miR-143 mimics/inhibitor and H/R challenge. The quantitative analysis of protein bands gray intensity was displayed in the histogram (right). The asterisks show difference significant as *p < .05, **p < .01, ***p < .001.

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Fig. 4. MiR-143 is a directly interacted target of lncUCA1. (A) Schematic picture of the predicted interaction sites between lncUCA1 and miR-143. (B) Luciferase activity analysis in HEK-293 cells co-transfected with reporter plasmid inserted with wild-type or mutated lncUCA1 sequences and miR-143 mimics or miR-NC. (C) The gene expression analysis of lncUCA1 and miR-143 by Q-PCR in cardiomyocytes treated with lncUCA1 overexpressed- (left) or shRNA-lentivirus (right). (D) qRTPCR analysis of the expression of lncUCA1 and miR-143 in cardiomyocytes transfected with miR-143 mimics (left) or inhibitor (right). The asterisks show difference significant as *p < .05, **p < .01.

silencing of lncUCA1 induced miR-143 expression (Fig. 4C); as well, the miR-143 level also negative regulated the lncUCA1 level (Fig. 4D), suggesting miR-143 may induce lncUCA1 degradation by post-transcription silencing. These results indicated that miR-143 was a directly interacted target of lncUCA1.

apoptosis was verified in this study. MDM2, a direct target of miR-143, was markedly inhibited by miR-143, but dramatically upregulated when miR-143 was silenced by inhibitor in cardiomyocytes (Fig. 3A). The activation of p53 by phosphorylation, which may be suppressed by MDM2, was promoted by miR-143 mimics treatment but inhibited when miR-143 was silenced by inhibitor in cardiomyocytes (Fig. 3A). As expected, miR-143 observably promoted H/R-induced cardiomyocyte apoptosis by overexpression, while reduced H/R-induced cardiomyocyte apoptosis when miR-143 was silenced by inhibitor (Fig. 3B). The expression of pro-apoptosis proteins Bax and Caspase-3 increased significantly accompanied by miR-143 overexpression but decreased with miR-143 silencing in H/R-treated cardiomyocytes, and the expression of anti-apoptosis protein Bcl-2 showed reversed phenotype (Fig. 3C). All these results indicated that miR-143 contributed to cardiomyocyte apoptosis induced by H/R through targeting MDM2 and activating p53 signal pathway.

3.5. LncUCA1 protected cardiomyocytes against H/R induced apoptosis by targeting miR-143 and MDM2/p53/Bcl-2 signal pathway To further confirm the regulation function of lncUCA1 on miR-143 and its downstream signaling pathway, lncUCA1 recombinant lentivirus were transfected into cardiomyocytes before H/R treatment. Results showed Lv-lncUCA1 prevented the increase of miR-143 expression and activation of p53 signaling pathway induced by H/R but activated the Akt/mTOR signaling pathway which inhibited cardiomyocytes apoptosis; the Lv-shlncUCA1 played adverse effects (Fig. 5A and B). To further verify the participation of miR-143/MDM2/p53 axis in the lncUCA1 function of apoptosis inhibition, combined treatment of Lv-lncUCA1 with miR-143 mimics or RITA (a p53 activator) was performed in cardiomyocytes before H/R treatment. Q-PCR analysis displayed that co-overexpression of miR-143 reduced the lncUCA1 level, and RITA increased miR-143 expression but also reduced the lncUCA1 level in cardiomyocytes co-treated with Lv-UCA1 and H/R challenge (Fig. 5C). As expected, co-overexpression of miR-143 or p53 activator RITA treatment activated p53 signaling by suppressing MDM2 and inactivated Akt/mTOR signaling, resulting in pro-apoptosis marker proteins Bax and Caspase-3 upregulation and anti-apoptosis marker protein Bcl-2 downregulation, which reversed the effect of lncUCA1 in cardiomyocytes co-treated with Lv-UCA1 and H/R challenge (Fig. 5D). All these results indicated that lncUCA1 might function as a cardioprotective factor through apoptosis inhibition by targeting miR-143 and MDM2/p53/Bcl-2 signal axis.

3.4. MiR-143 is a directly interacted target of lncUCA1 Three potential binding sites (Seed1、Seed2、Seed3) between lncUCA1 and miR-143 were predicted by softwares miRanda, RNAhybrid and RIsearch, and mutations were introduced into these sites (in bold) to create miRNA insensitive lncRNA sequences (Fig. 4A). The dual-luciferase reporter assays displayed that miR-143 reduced the luciferase activity with wild-type of lncUCA1, but there was no significant effect on the luciferase activity with Seed2-mutant lncUCA1 except the Seed1 and Seed3 (Fig. 4B), indicating the actual interaction site of lncUCA1 and miR-143 is the Seed2. To verify the reciprocal effects of lncUCA1 and miR-143 on expression regulation, lncUCA1 recombinant lentivirus and miR-143 mimics/inhibitor were transfected into cardiomyocytes respectively. Q-PCR analysis showed that overexpression of lncUCA1 dramatically suppressed miR-143 expression but 5

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Fig. 5. LncUCA1 protected cardiomyocytes against H/R induced apoptosis by targeting miR-143 and MDM2/p53/Bcl-2 signal pathway. (A) qRT-PCR analysis of the expression of miR-143 in cardiomyocytes co-treated with lncUCA1-recombinant lentivirus and H/R challenge. (B) Immunobloting analysis of MDM2, p53, phosphp53, Akt, phosph-Akt and mTOR in cardiomyocytes co-treated with lncUCA1-recombinant lentivirus and H/R challenge. (C) qRT-PCR analysis of the expression of lncUCA1 and miR-143 in cardiomyocytes co-treated with lncUCA1 overexpressed-lentivirus and H/R challenge, accompanied by miR-143 mimics or RITA administration. (D) Immunobloting analysis of MDM2/p53, Akt/mTOR and apoptosis marker proteins Bcl/Bax/Caspase-3 in cardiomyocytes co-treated with lncUCA1 overexpressed-lentivirus and H/R challenge, accompanied by miR-143 mimics or RITA administration. The asterisks show difference significant as **p < .01, ***p < .001.

4. Discussion

proliferation in vitro and xenograft tumor growth in vivo [27]. Moreover, overexpression of miR-143 arrested hemangioma-derived endothelial cells (HemECs) at the G0/G1 phase and promoted caspase-3dependent apoptosis [28]. More researches showed that miR-143 contributed to apoptosis via targeting MDM2 and indirectly activating the p53 pathway, which in turn activated the transcription of miR-143, thereby generating a short miR-143-MDM2-p53 feedback loop [29–31]. In this study, our findings suggested that miR-143 activated p53 phosphorylation by inhibit the expression of MDM2 in cardiomyocytes, but silencing miR-143 by inhibitor reversed the effects. H/R stimulates significant increase of miR-143 level and cardiomyocyte apoptosis, but silencing miR-143 by inhibitor reduced remarkably H/R induced cardiomyocyte apoptosis. All the results indicated that H/R induced cardiomyocyte apoptosis via upregulating miR-143 expression and modulated MDM2/p53/Bcl-2 signal pathway. One of the regulatory mechanisms of lncRNA is as the molecular sponge for miRNA, a competing endogenous RNA (ceRNA) mechanism, which has been extensively studied in heart diseases, including myocardial infarction [4,5,24]. CARL, a cardiac apoptosis-related lncRNA, is able to upregulate the level of prohibitin 2 (PHB2) by competitively binding with PHB2 upstream negative regulator miR-539, thereby inhibiting cardiomyocyte apoptosis [16]. HOTAIR dramatically limited hypoxia-induced myocyte apoptosis partly based on the negative

Increasing evidence suggests that lncRNAs may function as protective factors or risk factors of heart disease, such as lncRNA HOTAIR and Myheart [18–20]. In normal states, lncUCA1 is only expressed in heart and spleen of postnatal development, as well as higher level in adult heart [12,21], indicating that lncUCA1 might be an essential factor for cardiac function. Studies have shown that lncUCA1 was downregulated both in AMI patients and MIRI rat models [14,15]. Furthermore, lncUCA1 was proved to promote H2O2-induced cardiomyocyte apoptosis partially through stimulation of p27 protein expression [15]. Our present study also demonstrated that the level of lncUCA1 decreased significantly in cardiomyocytes suffering H/R treatment and cell apoptosis increased remarkably, which was reversed by lncUCA1overexpression but aggravated by lncUCA1 knockdown, suggesting that lncUCA1 might play a cardioprotective role in H/R-induced cardiomyocyte apoptosis. LncRNA is a regulatory RNA and able to regulate many genes in a post-transcriptional manner, including the microRNA (miRNA) [16,22–24]. MiR-143 was identified to be upregulated in myocardial infarction, serving as a pro-apoptosis miRNA on cardiomyocytes [25,26]. By directly targeting KRAS, miR-143 was identified as a tumor suppressor in nasopharyngeal carcinoma to suppress cell viability and 6

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regulation of miR-1 [18]. H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated upregulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK [22]. The direct interactions between lncUCA1 and miR-143 have been identified in breast cancer by RNA immunoprecipitation (RIP)/RT-PCR assay [32]. And there were multiple binding sites between lncUCA1 and miR-143 by prediction software (miRanda, RNAhybrid and RIsearch). In our research, we have verified one of the three predicted binding sites by dual-luciferase reporter assay. Overexpression of lncUCA1 dramatically suppressed miR-143 expression but silencing of lncUCA1 induced miR143 expression in cardiomyocytes. Analogously, the miR-143 level also negative regulated the lncUCA1 level, suggesting miR-143 may induce lncUCA1 degradation by post-transcription silencing. What's more, further functional research in H/R induced cardiomyocytes showed that lncUCA1 lowered miR-143 expression and affected its downstream regulation to MDM2/p53 pathway, as well as the p53 downstream signaling Akt/mTOR pathway [33], thereby suppressing H/R induced cardiomyocytes apoptosis and injury. In addition, co-overexpression of miR-143 or p53 activator RITA treatment reversed the effect of lncUCA1, indicated that lncUCA1 might functioned as a cardioprotective factor through apoptosis inhibition by targeting miR-143 and MDM2/p53/Bcl-2 signal axis. In conclusion, our findings recovered a novel mechanism of lncUCA1 in regulating H/R-induced cardiomyocyte apoptosis by suppressing miR-143 and modulated the following downstream MDM2/ p53 signaling pathway, providing new promising biomarkers or therapeutic targets aiming at myocardial infarction and heart failure.

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Acknowledgements

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We appreciate the editors and reviewers for their kindly comments and suggestions.

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Conflict

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All authors declare no conflict of interest.

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