RETRACTED ARTICLE: LncRNA TUG1 protects against cardiomyocyte ischaemia reperfusion injury by inhibiting HMGB1

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Hanyu Shi, Zhenhua Dong & Haiqing Gao (2019) LncRNA TUG1 protects against cardiomyocyte ischaemia reperfusion injury by inhibiting HMGB1. Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 3511–3516, DOI: 10.1080/21691401.2018.1556214 It has come to our attention that the full authorship list and affiliations for this manuscript were changed after the article was submitted. We have contacted the authors for an explanation, but we have not received a response within the requested timeframe. As determining authorship is core to the integrity of published work, we are therefore retracting the article. The authors listed in this publication have been sent notification. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.


Introduction
Myocardial ischaemic reperfusion injury (MIRI) is a phenomenon that myocardial ischaemic injury is aggravated when ischaemic myocardial restores blood [1]. The incidence of coronary heart disease with acute myocardial infarction is increased worldwide now [2]. In the process of blood flow reperfusion, however, it leads to a large number of inflammatory cells aggregation in the myocardium of original ischaemia, serious damage to vascular endothelial function, obvious metabolic dysfunction, arrhythmia, myocardial apoptosis [3][4][5]. It is the main cause of serious cardiac complications even death after coronary revascularization and heart transplantation [6,7]. Therefore, it has important clinical significance of reducing the injury of myocardial tissue after myocardial ischaemia and reperfusion, which can significantly reduce post-operative complications and mortality.
Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides, and exert their physiological and pathological functions through their interactions with genomic DNA, miRNAs, mRNAs and proteins [17,18]. At present, several studies have revealed the important roles of dysregulated lncRNA profiles in the pathogenesis of ischaemic injury in various organs including liver, heart and brain [19,20]. For instance, suppression of lncRNA KCNQ1QT1 may prevent myocardial I/R injury following acute myocardial infarction via regulating AdipoR1 and involving in p38 MAPK/NF-jB signal pathway [21]. LncRNA ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX [22]. LncRNA MALAT1 was expressed at a high level in patients with acute myocardial infraction and was closely associated with the pathogenesis of myocardial I/R injury [23].
Taurine-upregulated gene 1 (TUG1) was firstly reported to be upregulated in exposure to the treatment of taurine in mouse retinal cells [24]. TUG1 has been proved to act as a tumour suppressor or oncogene in various cancers [25][26][27]. However, whether dysregulation of lncRNA TUG1 has protective effects against myocardial ischaemia/reperfusion injury following acute myocardial infraction has not been fully investigated.
In present study, we used the mouse cardiomyocytes HL-1 cells under condition of oxygen glucose deprivation followed by reperfusion (OGD/R) to induce myocardial I/R injury in vitro. The effects of up-regulation or down-regulation of TUG1 on cell viability, inflammatory response and cell apoptosis were investigated. Moreover, the regulatory relationship between TUG1 and HMGB1 expression was also determined. Our study aimed to investigate the protective effects of TUG1 on the cardiomyocyte ischemia reperfusion injury and its underlying molecular mechanisms.

Cell lines
The mouse cardiomyocytes HL-1 cell line was purchased from ATCC. HL-1 cells were cultured in Claycomb medium containing 10% foetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 0.1 mM norepinephrine and 2 mM L-Glutamine at 37 C in an incubator with 95% air and 5% CO 2 .
Myocardial ischemia reperfusion injury model HL-1 cells processed a condition of oxygen glucose deprivation followed by reperfusion (OGD/R) to induce myocardial ischaemia reperfusion injury model in vitro. In brief, the cells were incubated for 24 h in 96-well plates, then cultured with glucose-free Claycomb medium and incubated in an oxygenfree atmosphere (95% N 2 and 5% CO 2 , 37 C) for 4 h. Following, the cells were incubated in normal culture medium (4.5 mg/mL glucose) and normal atmosphere (95% air and 5% CO 2 , 37 C) for another 24 h.

Cell transfection
Vector pc-TUG1 for overexpression of TUG1 was constructed by inserting the coding oligonucleotides of TUG1 into pcDNA3.1 vector (Invitrogen, Shanghai, China). pcDNA3.1 vector (pc-NC) was considered as the control. Small interference RNAs (siRNAs) targeting TUG1 (si-TUG1) and its control siRNAs (si-NC) were designed and synthesized by Invitrogen (Shanghai, China). OGD/R-induced I/R injury model HL-1 cells were cultured in the plates for 24 h and transfected with pc-TUG1, pc-NC, si-TUG, and si-NC using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The cells were incubated 48 h after transfection before next experiments.

Cell treatment
To determine the relationship between TUG1 and HMGB1 protein in preventing OGD/R-induced myocardial injury, the OGD/R-induced myocardial HL-1 cells were exposed to 10 lM of HMGB1 inhibitor sodium butyrate (Aladdin, Shanghai, China) during the OGD/R procedures.

MTT assay
Cells were seeded into a 96-well plate with a density of 1 Â 10 4 cells/mL. Cell viability was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay. After 24, 48, 72 and 96 h of transfection, 10 lL MTT solution (5 mg/mL, Beyotime, Shanghai, China) was added into each well and cultured for 4 h. The absorbance at 490 nm was measured after 150 lL dimethylsulphoxide (DMSO, Sigma) added and shaken for 10 min. The curve of cell proliferation was then drawn and the proliferation efficiency was examined. The experiments were repeated three times independently.

Cell apoptosis assay
Cell apoptosis was determined using an Annexin-V fluorescein isothiocyanate and propidium iodide (FITC/PI) apoptosis detection kit (Life Technologies, Waltham, MA) with flow cytometry in accordance with the manufacturer's instruction. Briefly, 1 Â 10 6 cells were harvested after 48 h transfection and stained using Annexin-V FITC/PI. Cell samples were analysed using flow cytometry and a FACScan within 1 h.

Western blotting
Protein expression levels were analysed by Western blot. Briefly, the cells were harvested and lysed on ice for 30 min in buffer. After centrifugation, the concentrations of protein were determined and separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Then, protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated into rabbit anti-HMGB1

Statistical analysis
All statistical analysis was carried out using the SPSS 18.0 software (SPSS, Inc., Chicago, IL). All values are expressed as the mean ± standard deviation from at least three repeated individual experiments for each group. The significant differences were analysed using Student's t-test or v 2 analysis for difference between two groups, and one-way ANOVA followed by Kruskal-Wallis H test for multiple comparison. Differences were considered significant when p < .05.

Knock down of TUG1 protected OGD/R-induced myocardial I/R injury
As shown in Figure 1(a), the lncRNA TUG1 was significantly overexpression in OGD/R-induced myocardial HL-1 cells (p < .05). The level of TUG1 expression was significantly decreased in si-TUG1 transfected group and significantly increased in pc-TUG1 transfected group (Figure 1(a), p <.05), compared to the control group. In addition, the cell viability significantly decreased after OGD/R or TUG1 overexpression treatment compared to the control cells, which reversed by TUG1 knock down. These data indicated that suppression of TUG1 could prevent OGD/R-induced myocardial I/R injury.

Knock down of TUG1 inhibited inflammation in OGD/Rinduced myocardial HL-1 cells
As shown in Figure 2, the expression of TNF-a and IFN-c were significantly up-regulated after OGD/R treatment compared to the control group (p < .05), while inhibited expression in the TUG1 knock down group (p < .05). These suggested that overexpression of TUG1-induced inflammation in OGD/R-induced myocardial HL-1 cells.

Knock down of TUG1 inhibited OGD/R-induced myocardial HL-1 cells apoptosis
As shown in Figure 3(a), overexpression of TUG1 induced the OGD/R-induced myocardial HL-1 cells apoptosis, which could be inhibited after si-TUG1 transfected (p < .05). Furthermore, the apoptosis-related proteins detection showed that Bcl-2 and pro-caspase-3 were significantly decreased and Bax and cleaved caspase-3 were significantly increased in OGD/Rinduced myocardial HL-1 cells (Figure 3(b), p <.05). In addition, the expression changes of these apoptosis-related proteins were significantly reversed after knock down of TUG1 (Figure 3(b), p <.05). Those suggested that suppression of TUG1 inhibited the apoptosis of OGD/R-induced myocardial HL-1 cells.

Knock down of TUG1 protected OGD/R-induced myocardial I/R injury by inhibiting HMGB1 expression
As shown in Figure 4(a), HMGB1 was significantly up-regulated in OGD/R-induced HL-1 cells compared to the control group (p < .05), while significantly inhibited after suppression of TUG1 (p < .05). To further determine whether knock down of TUG1 protected OGD/R-induced myocardial I/R injury through regulating HMGB1 expression. The HMGB1 inhibitor was used to treat OGD/R-induced myocardial HL-1 cells. The results showed that the effects of TUG1 regulated inflammatory factors expression in OGD/R-induced myocardial HL-1 cells were reversed by HMGB1 inhibitor (Figure 4(b), p <.05).
In addition, the percentage of apoptosis cells were significantly reversed after HMGB1 inhibitor treatment (Figure 4(c), p <.05). Those suggested that knock down of TUG1 protected OGD/R-induced myocardial I/R injury by inhibiting HMGB1 expression.

Discussion
Myocardial muscle ischaemia reperfusion injury is a common pathophysiological change in clinical vascular surgery. All kinds of cardiovascular surgery, especially the treatment of myocardial infarction, may be accompanied by myocardial ischemia reperfusion [28][29][30]. I/R is a double-edged sword.
On the one hand, it can solve the phenomenon of the

R E T R
A C T E D function of cardiomyocytes. On the other hand, it may lead to the occurrence of reperfusion injury, which called MIRI [31][32][33][34]. MIRI could cause changes in the cellular environment, such as oxidative stress, inflammatory response, and cellular Ca 2þ overload [35]. Those could lead to cell apoptosis or necrosis, and myocardial dysfunction is inevitable [36]. In the present study, we explored function and underlying mechanism of lncRNA TUG1 in the cardiomyocyte ischaemia reperfusion injury. Our study determined that TUG1 expression was markedly overexpression in OGD/R-induced myocardial cell lines. Moreover, TUG1 knockdown inhibited inflammation, apoptosis in OGD/R-induced myocardial cell lines. Furthermore, our findings suggest that TUG1 knockdown protect OGD/R-induced myocardial I/R injury by inhibiting HMGB1 expression.
More and more evidence have suggested that lncRNAs could be novel biomarkers for diagnosis and treatment of various diseases [37][38][39][40]. Yu et al. reported that lncRNA MALAT1 may increase cardiomyocyte autophagy and myocardial injury during I/R by negatively regulating miR-204 expression [20]. Li et al. reported that suppression of KCNQ1OT1 may prevent myocardial I/R injury following acute myocardial infarction via regulating AdipoR1 and involving in p38 MAPK/NF-jB signal pathway [18]. Yu et al. reported that lncRNA UCA1 modulates cardiomyocyte apoptosis by targeting miR-143 in myocardial ischemia-reperfusion injury [41]. LncRNA TUG1 was overexpressed in several kinds of cancer tissues and could function as an oncogene or tumour suppressor in different cancers [42][43][44][45][46]. However, its function in myocardial injury during I/R has not investigated yet. In the present study, it is firstly reported the lncRNA TUG1 overexpression in OGD/R-induced myocardial cell lines and TUG1 knockdown protect OGD/R-induced myocardial I/R injury by inhibiting HMGB1 expression.
In the early stage of reperfusion, IRI can induce nuclear transfer of nuclear factor-jB and causes serum pro-inflammatory factors such as TNF-a, IL-6 increasing [47,48]. In the present study, HMGB1, TNF-a and IL-6 could regulate inflammation response and cell damage in the early stage of MIRI injury. Simultaneously, HMGB1 could induce TNF-a and IL-6 release and further aggravation of myocardial IRI [49]. Zhao et al. reported that down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 release and protects against liver IRI, which is helpful for better understanding the role of HMGB1 in organ IRI [50]. Li et al. reported that miR-26a inhibited HMGB1 expression and attenuated cardiac ischaemia-reperfusion injury [51]. However, how it is regulated in myocardial ischaemia-reperfusion injury is not quite clear. In the present study, HMGB1 was significantly up-regulated in OGD/R-induced HL-1 cells. HMGB1 inhibitor could reverse the TUG1 regulated inflammatory factor expression, apoptosis. Our study presented a positive correlation between TUG1 and HMGB1 expression.
However, the molecule mechanism of lncRNA TUG1 inhibited HMGB1 expression in OGD/R-induced myocardial cells lines could not be well documented in the current study due to its limit of retrospective cohort. Additional in vivo experiments and clinical trials are warranted to justify this approach for lncRNA TUG1 target therapy in the future.
In conclusion, our findings determine that suppression of lncRNA TUG1 may prevent myocardial I/R injury following acute myocardial infarction via inhibiting HMGB1 expression. TUG1 may serve as a potential biomarker or therapeutic target for acute myocardial infraction. Further studies are still needed to verify our findings and hypothesis.

Disclosure statement
No potential conflict of interest was reported by the authors.