Open Access

Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324

  • Authors:
    • Jiong Li
    • Jing Xie
    • Yan-Zhen Wang
    • Yi-Rong Gan
    • Ling Wei
    • Guan-Waner Ding
    • Yan-Hong Ding
    • Ding-Xiong Xie
  • View Affiliations

  • Published online on: December 3, 2020     https://doi.org/10.3892/mmr.2020.11755
  • Article Number: 116
  • Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Endoplasmic reticulum stress (ERS) contributes to the pathogenesis of myocardial ischemia/reperfusion injury and myocardial infarction (MI). Long non-coding RNAs (lncRNAs) serve an important role in cardiovascular diseases, and lncRNA discrimination antagonizing non-protein coding RNA (Dancr) alleviates cardiomyocyte damage. microRNA (miR)-6324 was upregulated in MI model rats and was predicted to bind to Dancr. The present study aimed to investigate the role of Dancr in ERS-induced cardiomyocytes and the potential underlying mechanisms. Tunicamycin (Tm) was used to induce ERS. Cell viability, apoptosis and levels of associated proteins, ERS and autophagy in Dancr-overexpression H9C2 cells and miR-6234 mimic-transfected H9C2 cells were assessed using Cell Counting Kit-8, TUNEL staining and western blot assay, respectively. The results suggested that Dancr expression levels and cell viability were downregulated by Tm in a concentration-dependent manner compared with the control group. Tm induced apoptosis, ERS and autophagy, as indicated by an increased ratio of apoptotic cells, increased expression levels of Bax, cleaved (c)-caspase-3/9, glucose-regulated protein 78 kDa (GRP78), phosphorylated (p)-inositol-requiring enzyme-1α (IRE1α), spliced X-box-binding protein 1 (Xbp1s), IRE1α, activating transcription factor (ATF)6, ATF4, Beclin 1 and microtubule associated protein 1 light chain 3α (LC3)II/I, and decreased expression levels of Bcl-2, unspliced Xbp1 (Xbp1u) and p62 in the Tm group compared with the control group. Moreover, the results indicated that compared with the Tm + overexpression (Oe)-negative control (NC) group, the Tm + Oe-Dancr group displayed decreased apoptosis, but enhanced ERS and autophagy to restore cellular homeostasis. Compared with the Tm + Oe-NC group, the Tm + Oe-Dancr group decreased the ratio of apoptotic cells, decreased expression levels of Bax, c-caspase-3/9 and Xbp1u, and increased expression levels of Bcl-2, p-IRE1α, Xbp1s, Beclin 1 and LC3II/I. Dancr overexpression also significantly downregulated miR-6324 expression compared with Oe-NC. The dual-luciferase reporter assay further indicated an interaction between Dancr and miR-6324. In addition, miR-6324 mimic partially reversed the effects of Dancr overexpression on Tm-induced apoptosis, ERS and autophagy. In conclusion, lncRNA Dancr overexpression protected cardiomyocytes against ERS injury via sponging miR-6324, thus inhibiting apoptosis, enhancing autophagy and restoring ER homeostasis.

Introduction

With social and economic development and the aging population, the occurrence of cardiovascular events is increasing each year (1). Among cardiovascular diseases, acute myocardial infarction (AMI), which is associated with high lethality and high disability, has become the leading cause of human mortality globally (2). AMI refers to myocardial necrosis caused by acute or continuous ischemia and hypoxia in the coronary arteries (3). Myocardial ischemia is a pathological state in which blood perfusion of the heart is decreased, resulting in decreased oxygen supply to the heart and abnormal energy metabolism of the myocardial cells, which cannot support the normal function of the heart (4). Persistent and acute ischemia of the heart can develop into AMI (5).

At present, the treatment strategies for AMI primarily include thrombolysis, percutaneous coronary intervention and surgical bypass surgery. The primary purpose of the therapeutic strategies is to restore the blood supply to the heart, rescue myocardial cells in the infarction area and prevent further damage caused by ischemia (6). However, the current treatment strategies have limited therapeutic effects on damaged cardiomyocytes, as cardiomyocytes in adults cannot be naturally regenerated once they are lost (7). Therefore, rescuing myocardial cells at the near-death state in the marginal area of MI by investigating the function of certain specific long non-coding RNAs (lncRNAs) or proteins is a novel research topic for treating AMI.

lncRNAs are transcripts >200 nucleotides in length, lacking a specific complete open reading frame and protein coding functions (8). lncRNAs function as crucial regulators via regulating gene expression at both the transcriptional and post-transcriptional levels (9). lncRNAs can regulate microRNAs (miRNAs/miRs) by serving as miRNA sponges, adsorbing the corresponding miRNA and exerting transcriptional regulation (10). A number of studies have demonstrated that lncRNAs serve important roles in the occurrence and development of human diseases, including cardiovascular diseases (11,12).

lncRNA discrimination antagonizing non-protein coding RNA (Dancr) encodes human chromosome 4q12, and was first identified as an epidermal cell differentiation suppressor (13). Further investigation revealed that Dancr functions as an oncogene in various types of cancer, including hepatocellular carcinoma (14), non-small cell lung carcinoma (15,16) and osteosarcoma (17). Recently, Dancr has been reported to alleviate hypoxia-induced H9c2 cardiomyocyte damage by upregulating hypoxia inducible factor-1α (18). In addition, Dancr decreased hypoxia- and hypoglycemia-induced damage of cerebral microvascular endothelial cells via regulating miR-33a-5p/spliced X-box-binding protein 1 (Xbp1s) (19). However, to the best of our knowledge, the effect of Dancr in myocardial ischemia and myocardial infarction has not been previously reported.

In the case of MI/reperfusion (R), endoplasmic reticulum (ER) homeostasis is destroyed, resulting in the accumulation of a large number of unfolded or misfolded proteins in the ER, triggering the unfolded protein response (UPR) and ER stress (ERS) (20). Early ERS exerts a compensatory protective effect, but excessive ERS is involved in the pathophysiological process of various cardiovascular diseases (21). Tunicamycin (Tm) is a commonly used ERS inducer (22). Previous studies have demonstrated that low doses of Tm can produce moderate ERS, which displays a certain protective effect on MI/R (23,24). Moderate ERS can also induce autophagy to help the degradation of unfolded or misfolded proteins, thus alleviating ERS (25). However, excessive ERS may initiate the apoptosis reaction and inflammatory pathways, ultimately participating in the deterioration of cardiovascular disease (26). ERS in response to various adverse stimuli has been detected and has been reported to be associated with the pathogenesis of MI/R injury, myocardial hypertrophy, ischemic cardiomyopathy, diabetic cardiomyopathy and cardiac fibrosis (27). For example, miR-711 mimic could induce cardiomyocyte apoptosis after ERS-induced MI via upregulating Xbp1 (28). Xu et al (29) demonstrated that inhibition of ERS and the cell apoptosis signaling pathway could protect cardiomyocytes against MI-induced injury.

Autophagy is an important metabolic process that degrades senescent or damaged proteins and organelles into amino groups (30). Autophagy is activated in response to nutritional deficiencies or metabolic stress to maintain tissue function and homeostasis (31). Basic autophagy has been reported to be essential for maintaining normal heart function. Meanwhile, under ischemic stress, autophagy is activated to protect cardiomyocytes from ischemia or I/R injury (32). The beneficial role of autophagy in AMI in the alleviation of MI under ischemic and ischemia/R injuries has been extensively reported (33,34). Therefore, taking advantage of autophagy provides a potential strategy for the development of novel drugs or therapies for AMI (35).

The aforementioned studies indicated that the moderate enhancement of ERS and autophagy, together with the repression of excessive ERS and ERS-mediated apoptosis might serve as a valuable therapeutic strategy for relieving MI-induced injury. The present study aimed to investigate the roles and molecular mechanism underlying Dancr in ERS-induced cardiomyocytes to provide a novel target for the diagnosis and therapy of AMI.

Materials and methods

Cell culture and treatment

The H9c2 rat embryonic cardiomyocyte cell line (American Type Culture Collection) was cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 units/ml penicillin at 37°C in a humidified atmosphere of 5% CO2. The medium was replaced every other day. At 70–80% confluence, cells were digested with trypsin and EDTA.

Tm (MedChemExpress) was utilized to induce ERS. H9C2 cells were treated Tm (0.1, 0.5, 2.5 or 12.5 µM) for 6 h at 37°C and control cells were cultured in normal medium (36).

Cell transfection

Prior to transfection, the medium was replaced with serum- and antibiotic-free DMEM. To overexpress lncRNA Dancr, the recombinant full-length rat Dancr cDNA [overexpression (Oe)-Dancr] was cloned into the pcDNA3.1 vector (Thermo Fisher Scientific, Inc.). The pcDNA3.1 empty vector was used as a negative control (NC; Oe-NC). H9C2 cells (60-70% confluence) were transfected with oe-Dancr, oe-NC, miR-6324 mimic (5′-AGUAGGCCAGACAGCAAGC-3′; Sigma-Aldrich; Merck KGaA) or mimic-NC (5′-GGUUCGUACGUACACUGUUCA-3′; Sigma-Aldrich; Merck KGaA) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, Lipofectamine 2000 was mixed with 50 nM plasmids or mimics, added to the cells and incubated for 6 h at 37°C. Subsequently, cells were cultured in DMEM for 24 h and then used for subsequent experiments. Transfection efficiency was evaluated via reverse transcription-quantitative PCR (RT-qPCR).

Cell Counting Kit-8 (CCK-8)

The CCK-8 assay was performed to assess cell viability. H9c2 cells (1×104 cells/well) were cultured in 96-well plates and subjected to corresponding treatments. Subsequently, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and incubated at 37°C for 2 h in the dark. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader.

TUNEL staining

Apoptotic H9c2 cardiomyocytes were visualized by performing TUNEL staining (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturers protocol. Briefly, H9c2 cells were cultured on cover slips. After the corresponding treatment, cells were fixed with 4% neutral buffered formalin solution for 30 min at room temperature. Subsequently, 50 µl TUNEL reaction mixture was added and incubated for 1 h at 37°C. The nuclei were stained with DAPI (2 µg/ml) at room temperature for 5 min. After washing twice with PBS, images were captured from three fields of view using a fluorescence microscope (magnification, ×200).

Western blotting

Total protein was extracted from H9c2 cardiomyocytes using lysis buffer (Beyotime Institute of Biotechnology) containing a protease inhibitor and phosphatase inhibitor. After being quantified using a BCA kit (Beyotime Institute of Biotechnology), equal amounts of protein (50 µg) were separated via 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk at 37°C for 2 h. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies targeted against: Bcl-2 (Abcam; cat. no. ab32124; 1:1,000), Bax (Abcam; cat. no. ab32503; 1:10,000), cleaved (c)-caspase-9 (Abcam; cat. no. ab32539; 1:5,000), caspase-9 (Abcam; cat. no. ab184786; 1:1,000), c-caspase-3 (Abcam; cat. no. ab32042; 1:500), caspase-3 (Abcam; cat. no. ab13847; 1:500), glucose-regulated protein 78 kDa (GRP78; Abcam; cat. no. ab21685; 1:1,000), phosphorylated (p)-inositol-requiring enzyme-1 (IRE1)α (Abcam; cat. no. ab124945; 1:1,000), IRE1α (Abcam; cat. no. ab37073; 1:1,000), Xbp1s (ProteinTech Group, Inc.; cat. no. 24868-1-AP; 1:1,000), unspliced Xbp1 (Xbp1u; ProteinTech Group, Inc.; cat. no. 25997-1-AP; 1:1,000), activating transcription factor (ATF)6 (ProteinTech Group, Inc.; cat. no. 24169-1-AP; 1:2,000), ATF4 (ProteinTech Group, Inc.; cat. no. 10835-1-AP; 1:1,000), Beclin 1 (ProteinTech Group, Inc.; cat. no. 11306-1-AP; 1:10,000), microtubule associated protein 1 light chain 3α (LC3)II/I (ProteinTech Group, Inc.; cat. no. 14600-1-AP; 1:2,000), p62 (Abcam; cat. no. ab56416; 1:1,000) and GAPDH (Abcam; cat. no. ab8245; 1:10,000). Following primary incubation, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG secondary antibodies at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and detection system (Amersham; Cytiva). Protein expression levels were semi-quantified using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.) with GAPDH as the loading control.

RT-qPCR

Total RNA was extracted from H9C2 cardiomyocytes using an RNA isolation kit (Total RNA Extraction Reagent; Vazyme Biotech Co., Ltd.). Total RNA was reverse transcribed into cDNA using a reverse transcriptase (Vazyme Biotech Co., Ltd.). Subsequently, qPCR was performed using the CFX384 Real-Time System C1000 Thermocycler (Bio-Rad Laboratories, Inc.) and SYBR-Green ROX-mix (Vazyme Biotech Co., Ltd.). The following primers were used for qPCR: Dancr forward, 5′-CTCGGATAGAAGCGCAGGTT-3′ and reverse, 5′-AGGCAAGCGGGGTCATTAAA-3′; miR-6324 forward, 5′-ATAGCTGGGGTCAAGGTGCT-3′ and reverse, 5′-CTTGCTGTCTGGCCTACTGA-3′; GAPDH forward, 5′-TTGTGCAGTGCCAGCCTC-3′ and reverse, 5′-GGTAACCAGGCGTCCGATAC-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec then 40 cycles of 95°C for 5 sec and 60°C for 15 sec, followed by default of melt curve. miRNA and mRNA expression levels were quantified using the 2−∆∆Cq method (37) and normalized to the internal reference genes U6 and GAPDH, respectively.

Dual-luciferase reporter assay

The wild-type (WT) and mutated (MUT) Dancr fragments containing the miR-6324 binding sites were synthesized and inserted into the pmirGLO luciferase vector (Promega Corporation) to generate WT-pmirGLO and MUT-pmirGLO, respectively. For the promoter analysis, miR-6324 mimic or mimic NC were cloned into the pGL3 luciferase reporter (Promega Corporation). Cells were co-transfected with WT-pmirGLO or MUT-pmirGLO vectors and miR-6324 mimic or mimic NC. At 48 h post-transfection, luciferase activities were measured using a Dual-Luciferase reporter assay system (Promega Corporation) as previously described (38).

Statistical analysis

Data are presented as the mean ± standard deviation of from at least three independent experiments. Comparisons between two groups were analyzed using the Students t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukeys post hoc test. Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

lncRNA Dancr is downregulated upon Tm stimulation

The present study aimed to determine whether Dancr could regulate cardiomyocytes that underwent ERS. Following stimulation with different concentrations of the ERS agonist Tm (0.1, 0.5, 2.5 or 12.5 µM), Dancr expression levels and cell viability in H9C2 cells were measured. Compared with the control group, Tm decreased Dancr mRNA expression levels (Fig. 1A) and cell viability (Fig. 1B) in a concentration-dependent manner, suggesting a modulatory effect of Dancr on cardiomyocyte ERS. Based on the finding that 2.5 µM Tm resulted in a >50% reduction in Dancr expression but maintained cell viability at ≥50%, 2.5 µM Tm was selected for subsequent experiments (39).

Dancr overexpression enhances cell viability and decreases cell apoptosis in Tm-stimulated H9C2 cardiomyocytes

To determine the specific role of Dancr in ERS-induced cardiomyocyte injury, Dancr was overexpressed in H9C2 cells. The results demonstrated the successful overexpression of Dancr (Fig. 2A). Oe-Dancr-1 was selected to establish Dancr-overexpression H9C2 cells in subsequent experiments based on its higher efficacy compared with Oe-Dancr-2. Moreover, compared with the control group, 2.5 µM Tm treatment significantly decreased cell viability, markedly increased the ratio of apoptotic cells, significantly decreased the expression levels of the antiapoptosis protein Bcl-2, and significantly increased the expression levels of the proapoptosis proteins Bax and c-caspase-3/9 (Fig. 2B-D). However, following Tm stimulation, compared with Oe-NC-transfected cells, Oe-Dancr-transfected cells displayed significantly higher cell viability, a notably lower ratio of cell apoptosis, significantly increased expression levels of Bcl-2, and significantly decreased expression levels of Bax and c-caspase-3/9.

Dancr overexpression enhances ERS and autophagy in Tm-stimulated H9C2 cardiomyocytes

The present study analyzed the expression levels of proteins associated with ERS and autophagy. Compared with the control group, Tm treatment resulted in significantly increased expression levels of GRP78, p-IRE1α, Xbp1s, IRE1α, ATF6 and ATF4, significantly decreased expression levels of Xbp1u and no significant alterations to the p-IRE1α/IRE1α ratio, indicating the induction of ERS (Fig. 3A). Compared with the Tm + Oe-NC group, the Tm + Oe-Dancr group displayed significantly enhanced p-IRE1α, p-IRE1α/IRE1α and Xbp1s expression levels and significantly decreased Xbp1u expression levels, but there were no significant alterations to the expression levels of IRE1α, ATF6 or ATF4, suggesting that Dancr selectively promoted the phosphorylation of IRE1α and activated the IRE1α branch in the UPR. Moreover, compared with the control group, Tm treatment induced autophagy, as evidenced by significantly increased expression levels of Beclin 1 and LC3II/I, and significantly decreased expression levels of p62 (Fig. 3B). Compared with the Tm + Oe-NC group, the Tm + Oe-Dancr group displayed increased levels of autophagy, as demonstrated by significantly increased Beclin 1 and LC3II/I expression levels, and significantly lower p62 expression levels.

miR-6324 mimic partially reverses the effects of Dancr overexpression on Tm-induced apoptosis, ERS and autophagy

miR-6324 was predicted to bind to Dancr (Fig. 4A). To verify whether miR-6324 was the target of Dancr, the present study constructed two miR-6324 mimics. The results verified the successful transfection of the miR-6324 mimics (Fig. 4B). The direct interaction between Dancr and miR-6324 was assessed by performing a Dual-luciferase reporter assay (Fig. 4C). Moreover, Dancr overexpression significantly downregulated miR-6324 expression levels compared with Oe-NC (Fig. 4D). The results suggested that miR-6324 might participate in the actions of Dancr on ERS-induced cardiomyocyte injury.

To investigate the modulatory effects of miR-6324 on Dancr-mediated H9C2 ERS, Tm-treated and Dancr-overexpression H9C2 cells were transfected with miR-6324 mimic or mimic-NC. Compared with the Tm + Oe-Dancr + mimic NC group, the Tm + Oe-Dancr + miR-6324 mimic group displayed significantly decreased cell viability and markedly increased numbers of apoptotic cells (Fig. 5A and B). Furthermore, compared with the Tm + Oe-Dancr + mimic NC group, the Tm + Oe-Dancr + miR-6324 mimic group displayed significantly decreased Bcl-2 expression levels, and significantly increased Bax and c-caspase-3/9 expression levels (Fig. 5C).

The expression levels of proteins associated with ERS and autophagy were measured (Fig. 6). Oe-Dancr-mediated upregulation of GRP78, p-IRE1α, p-IRE1α/IRE1α, Xbp1s, Beclin 1 and LC3II/I expression levels was significantly reversed by miR-6324 mimic. Similarly, miR-6324 mimic partially recovered the expression levels of Xbp1u and p62, which were decreased by Tm and further decreased by Dancr overexpression combined with Tm treatment. The aforementioned results indicated that miR-6324 mimic partially abolished Dancr overexpression-mediated effects on Tm-induced apoptosis, ERS and autophagy.

Discussion

lncRNA Dancr has been reported to protect H9c2 cardiomyocytes against hypoxia-induced damage (18). The present study indicated that Dancr protected H9C2 cells against ERS-induced apoptosis in vitro via the selective activation of the IRE1α/XBP1 signaling pathway in the UPR, which suggested that Dancr provided cytoprotection in response to ERS.

When the ER senses a wide variety of perturbations, including UPR, the adaptive process of ERS occurs to maintain ER homeostasis and mitigate or eliminate the stress (22). There are three primary ER transmembrane stress sensors that initiate the UPR, including IRE1, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and ATF6, are maintained in an inactive state via binding to their luminal domains with the ER chaperone GRP78. However, in the presence of ERS, GRP78 is released from these complexes and recruited to misfolded proteins, leading to the activation of three distinct UPR branches (40). For instance, the IRE1 branch possesses endoribonuclease activity that splices mRNAs encoding Xbp1u to form mature Xbp1/Xbp1s mRNA, which is then translated to a potent transcription factor that controls the genes encoding proteins that target misfolded proteins for ubiquitination and ER-associated degradation (ERAD). Meanwhile, ATF6 cooperates with IRE1α for the induction of Xbp1 transcription. The proteins balance the unfolded protein/chaperone system to provide ER homeostasis. If the cell fails to recover from ERS, the UPR represses the adaptive response and triggers apoptosis. Notably, PERK stimulates the expression of the proapoptotic transcription factor C/EBP homologous protein via the mobilization of ATF4 (41). Thus, the ERS serves a dual role, transmitting both adaptive and apoptotic signals. In the present study, compared with the control group, Tm decreased cell viability and induced cell apoptosis, which could be explained by the occurrence of excessive ERS, as evidence by increased expression levels of GRP78, p-IRE1α, IRE1α, ATF6, ATF4 and Xbp1s, and decreased expression levels of Xbp1u. As previously described, if ERS is too severe or prolonged, the UPR initiates an apoptotic response (42).

Autophagy is essential for maintaining protein homeostasis and is an evolutionarily conserved protein degradation signaling pathway that removes damaged or expired proteins and organelles by chelating in autophagosomes, which subsequently undergo lysosomal degradation (43). Previous studies have demonstrated that the ERS response can activate the autophagy-lysosome signaling pathway, which serves a major role in the cardiac stress response (44,45). Autophagy functions as a cellular stress signaling pathway and can assist with the degradation of proteins to recover ER homeostasis (44). In the present study, compared with the control group, Tm treatment increased the expression levels of Beclin 1 and LC3II/I, and decreased the expression levels of p62, indicating the induction of autophagy in response to ERS.

lncRNAs have been identified as novel targets for the treatment of cardiovascular diseases. For example, lncRNA CDKN2B antisense RNA 1 has been identified as the most powerful predictor of atherosclerosis (46,47). Chi et al (47) revealed that the lncRNA myocardial infarction associated transcript 1 led to a decrease in the expression of inflammatory factors via inhibition of the NF-κB signaling pathway, thereby decreasing myocardial cell apoptosis and inflammatory cell infiltration to decrease AMI damage. To the best of our knowledge, the present study suggested the role of Dancr in cardiomyocyte ERS for the first time. Compared with the Tm + Oe-NC group, Dancr overexpression increased cell viability by decreasing cell apoptosis and promoting autophagy in Tm-treated H9C2 cardiomyocytes. Furthermore, compared with the Tm + Oe-NC group, Dancr overexpression enhanced p-IRE1α, p-IRE1α/IRE1α and Xbp1s expression levels, and decreased Xbp1u expression levels, but displayed no significant effects on IRE1α, ATF6 and ATF4 expression levels in Tm-treated H9C2 cardiomyocytes, suggesting that Dancr selectively activated the IRE1α branch in the UPR, thus promoting autophagy and ERAD, and ultimately alleviating ERS. It has been reported that enhancing autophagy could protect cardiomyocytes from ERS and apoptosis (48). However, whether the antiapoptotic effect of Dancr is dependent on the autophagy signaling pathway, the ERS signaling pathway or other mediators requires further investigation.

lncRNAs regulate the occurrence and development of human diseases, including cardiovascular diseases, primarily via sponging miRNAs (11). Our previous unpublished data demonstrated that miR-6324 was upregulated in MI model rats. In the present study, online analysis and luciferase reporter assays confirmed that miR-6324 interacted with lncRNA Dancr. Therefore, the present study hypothesized that Dancr displayed cardioprotective effects via sponging miR-6324 and inhibiting its expression. To verify the hypothesis, the present study co-transfected Tm-treated H9C2 cells with Oe-Dancr and miR-6324 mimic. miR-6324 mimic partially reversed Dancr overexpression-mediated effects on cell viability, cell apoptosis, ERS and autophagy. The results indicated that miR-6324 may serve as a downstream target miRNA of Dancr to rescue cell viability, inhibit apoptosis and promote autophagy, thereby relieving cardiomyocyte ERS. However, apoptosis and autophagy are double-edged swords, thus consistent with previous studies (20,24,25), the present study identified Dancr as a potential target to reduce apoptosis and ERS, while enhancing autophagy in Tm-induced H9C2 cardiomyocytes. The enhanced cell viability indicated the protective effect of Dancr, but in vivo studies are required to verify the results of the present study. Moreover, as miR-6324 mimic did not completely reverse the effects of Dancr, other downstream targets of Dancr may exist and should be investigated in future studies.

In summary, the present study provided evidence that lncRNA Dancr sponged miR-6324, selectively activated the IRE1α/Xbp1 signaling pathway of autophagy, repressed apoptosis and enhanced autophagy, leading to amelioration of ERS. The actions of Dancr identified in the present study suggested its potential for the treatment of cardiovascular diseases.

Acknowledgements

Not applicable.

Funding

This study was supported by the Lanzhou Talent Project for Innovation and Entrepreneurship (grant no. 2015-RC-12) and the Health Science and Technology Development Project of Lanzhou (grant no. 2019-002).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors contributions

YHD, DXX, JL and JX conceived and designed the study. JL, JX, YZW, YRG, LW and GWD acquired and analyzed the data. JL and JX drafted the manuscript and figures. YHD and DXX revised the manuscript for critically important intellectual content. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Moran AE, Roth GA, Narula J and Mensah GA: 1990-2010 global cardiovascular disease atlas. Glob Heart. 9:3–16. 2014. View Article : Google Scholar : PubMed/NCBI

2 

Mehta LS, Beckie TM, DeVon HA, Grines CL, Krumholz HM, Johnson MN, Lindley KJ, Vaccarino V, Wang TY, Watson KE, et al American Heart Association Cardiovascular Disease in Women and Special Populations Committee of the Council on Clinical Cardiology, Council on Epidemiology and Preventionm Council on Cardiovascular and Stroke Nursing, Council on Quality of Care and Outcomes Research, : Acute Myocardial Infarction in Women: A Scientific Statement From the American Heart Association. Circulation. 133:916–947. 2016. View Article : Google Scholar : PubMed/NCBI

3 

Reed GW, Rossi JE and Cannon CP: Acute myocardial infarction. Lancet. 389:197–210. 2017. View Article : Google Scholar : PubMed/NCBI

4 

Tamis-Holland JE, Jneid H, Reynolds HR, Agewall S, Brilakis ES, Brown TM, Lerman A, Cushman M, Kumbhani DJ, Arslanian-Engoren C, et al American Heart Association Interventional Cardiovascular Care Committee of the Council on Clinical Cardiology; Council on Cardiovascular and Stroke Nursing; Council on Epidemiology and Prevention; and Council on Quality of Care and Outcomes Research, : Contemporary Diagnosis and Management of Patients With Myocardial Infarction in the Absence of Obstructive Coronary Artery Disease: A Scientific Statement From the American Heart Association. Circulation. 139:e891–e908. 2019. View Article : Google Scholar : PubMed/NCBI

5 

Heusch G and Gersh BJ: The pathophysiology of acute myocardial infarction and strategies of protection beyond reperfusion: A continual challenge. Eur Heart J. 38:774–784. 2017.PubMed/NCBI

6 

Asaria P, Elliott P, Douglass M, Obermeyer Z, Soljak M, Majeed A and Ezzati M: Acute myocardial infarction hospital admissions and deaths in England: A national follow-back and follow-forward record-linkage study. Lancet Public Health. 2:e191–e201. 2017. View Article : Google Scholar : PubMed/NCBI

7 

Dall C, Khan M, Chen CA and Angelos MG: Oxygen cycling to improve survival of stem cells for myocardial repair: A review. Life Sci. 153:124–131. 2016. View Article : Google Scholar : PubMed/NCBI

8 

Jarroux J, Morillon A and Pinskaya M: History, Discovery, and Classification of lncRNAs. Adv Exp Med Biol. 1008:1–46. 2017. View Article : Google Scholar : PubMed/NCBI

9 

Deniz E and Erman B: Long noncoding RNA (lincRNA), a new paradigm in gene expression control. Funct Integr Genomics. 17:135–143. 2017. View Article : Google Scholar : PubMed/NCBI

10 

Zhao Z, Sun W, Guo Z, Zhang J, Yu H and Liu B: Mechanisms of lncRNA/microRNA interactions in angiogenesis. Life Sci. 254:1169002020. View Article : Google Scholar : PubMed/NCBI

11 

Sallam T, Sandhu J and Tontonoz P: Long noncoding RNA discovery in cardiovascular disease: Decoding Form to Function. Circ Res. 122:155–166. 2018. View Article : Google Scholar : PubMed/NCBI

12 

Ballantyne MD, McDonald RA and Baker AH: lncRNA/MicroRNA interactions in the vasculature. Clin Pharmacol Ther. 99:494–501. 2016. View Article : Google Scholar : PubMed/NCBI

13 

Thin KZ, Liu X, Feng X, Raveendran S and Tu JC: LncRNA-DANCR: A valuable cancer related long non-coding RNA for human cancers. Pathol Res Pract. 214:801–805. 2018. View Article : Google Scholar : PubMed/NCBI

14 

Yuan SX, Wang J, Yang F, Tao QF, Zhang J, Wang LL, Yang Y, Liu H, Wang ZG, Xu QG, et al: Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1. Hepatology. 63:499–511. 2016. View Article : Google Scholar : PubMed/NCBI

15 

Guo L, Gu J, Hou S, Liu D, Zhou M, Hua T, Zhang J, Ge Z and Xu J: Long non-coding RNA DANCR promotes the progression of non-small-cell lung cancer by inhibiting p21 expression. OncoTargets Ther. 12:135–146. 2018. View Article : Google Scholar

16 

Bai Y, Zhang G, Chu H, Li P and Li J: The positive feedback loop of lncRNA DANCR/miR-138/Sox4 facilitates malignancy in non-small cell lung cancer. Am J Cancer Res. 9:270–284. 2019.PubMed/NCBI

17 

Wang Y, Zeng X, Wang N, Zhao W, Zhang X, Teng S, Zhang Y and Lu Z: Long noncoding RNA DANCR, working as a competitive endogenous RNA, promotes ROCK1-mediated proliferation and metastasis via decoying of miR-335-5p and miR-1972 in osteosarcoma. Mol Cancer. 17:892018. View Article : Google Scholar : PubMed/NCBI

18 

Qiu L, Zhao Q, Dai L, Zhu A, Xu X, Zhao S and Chen J: Long non-coding RNA DANCR alleviates hypoxia-caused H9c2 cells damage through up regulation of HIF-1α. Artif Cells Nanomed Biotechnol. 48:533–541. 2020. View Article : Google Scholar : PubMed/NCBI

19 

Zhang M, Tang M, Wu Q, Wang Z, Chen Z, Ding H, Hu X, Lv X, Zhao S, Sun J, et al: LncRNA DANCR attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation through regulating of miR-33a-5p/XBP1s. Aging (Albany NY). 12:1778–1791. 2020. View Article : Google Scholar : PubMed/NCBI

20 

Zuo S, Kong D, Wang C, Liu J, Wang Y, Wan Q, Yan S, Zhang J, Tang J, Zhang Q, et al: CRTH2 promotes endoplasmic reticulum stress-induced cardiomyocyte apoptosis through m-calpain. EMBO Mol Med. 10:e82372018.simplehttps://doi.org/10.15252/emmm.201708237 View Article : Google Scholar : PubMed/NCBI

21 

Wang X, Xu L, Gillette TG, Jiang X and Wang ZV: The unfolded protein response in ischemic heart disease. J Mol Cell Cardiol. 117:19–25. 2018. View Article : Google Scholar : PubMed/NCBI

22 

Yan M, Shu S, Guo C, Tang C and Dong Z: Endoplasmic reticulum stress in ischemic and nephrotoxic acute kidney injury. Ann Med. 50:381–390. 2018. View Article : Google Scholar : PubMed/NCBI

23 

Yamamoto K and Ichikawa S: Tunicamycin: Chemical synthesis and biosynthesis. J Antibiot (Tokyo). 72:924–933. 2019. View Article : Google Scholar : PubMed/NCBI

24 

Wang S, Wang Z, Fan Q, Guo J, Galli G, Du G, Wang X and Xiao W: Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway. Br J Pharmacol. 173:2402–2418. 2016. View Article : Google Scholar : PubMed/NCBI

25 

Fernández A, Ordóñez R, Reiter RJ, González-Gallego J and Mauriz JL: Melatonin and endoplasmic reticulum stress: Relation to autophagy and apoptosis. J Pineal Res. 59:292–307. 2015. View Article : Google Scholar : PubMed/NCBI

26 

Hu J, Huang CX, Rao PP, Cao GQ, Zhang Y, Zhou JP, Zhu LY, Liu MX and Zhang GG: MicroRNA-155 inhibition attenuates endoplasmic reticulum stress-induced cardiomyocyte apoptosis following myocardial infarction via reducing macrophage inflammation. Eur J Pharmacol. 857:1724492019. View Article : Google Scholar : PubMed/NCBI

27 

Li X, Zhao J, Geng J, Chen F, Wei Z, Liu C, Zhang X, Li Q, Zhang J, Gao L, et al: Long non-coding RNA MEG3 knockdown attenuates endoplasmic reticulum stress-mediated apoptosis by targeting p53 following myocardial infarction. J Cell Mol Med. 23:8369–8380. 2019. View Article : Google Scholar : PubMed/NCBI

28 

Zhao N, Mi L, Zhang X, Xu M, Yu H, Liu Z, Liu X, Guan G, Gao W and Wang J: Enhanced MiR-711 transcription by PPARγ induces endoplasmic reticulum stress-mediated apoptosis targeting calnexin in rat cardiomyocytes after myocardial infarction. J Mol Cell Cardiol. 118:36–45. 2018. View Article : Google Scholar : PubMed/NCBI

29 

Xu Y, Wu L, Chen A, Xu C and Feng Q: Protective effects of olive leaf extract on acrolein-exacerbated myocardial infarction via an endoplasmic reticulum stress pathway. Int J Mol Sci. 19:4932018.simplehttps://doi.org/10.3390/ijms19020493 View Article : Google Scholar

30 

Cătană CS, Atanasov AG and Berindan-Neagoe I: Natural products with anti-aging potential: Affected targets and molecular mechanisms. Biotechnol Adv. 36:1649–1656. 2018. View Article : Google Scholar : PubMed/NCBI

31 

Dong Z, Chu G, Sima Y and Chen G: Djhsp90s are crucial regulators during planarian regeneration and tissue homeostasis. Biochem Biophys Res Commun. 498:723–728. 2018. View Article : Google Scholar : PubMed/NCBI

32 

Wang L, Li Y, Ning N, Wang J, Yan Z, Zhang S, Jiao X, Wang X and Liu H: Decreased autophagy induced by β1-adrenoceptor autoantibodies contributes to cardiomyocyte apoptosis. Cell Death Dis. 9:4062018. View Article : Google Scholar : PubMed/NCBI

33 

Aisa Z, Liao GC, Shen XL, Chen J, Li L and Jiang SB: Effect of autophagy on myocardial infarction and its mechanism. Eur Rev Med Pharmacol Sci. 21:3705–3713. 2017.PubMed/NCBI

34 

Kanamori H, Takemura G, Goto K, Maruyama R, Ono K, Nagao K, Tsujimoto A, Ogino A, Takeyama T, Kawaguchi T, et al: Autophagy limits acute myocardial infarction induced by permanent coronary artery occlusion. Am J Physiol Heart Circ Physiol. 300:H2261–H2271. 2011. View Article : Google Scholar : PubMed/NCBI

35 

Wu D, Zhang K and Hu P: The role of autophagy in acute myocardial infarction. Front Pharmacol. 10:5512019. View Article : Google Scholar : PubMed/NCBI

36 

Yu Y, Sun G, Luo Y, Wang M, Chen R, Zhang J, Ai Q, Xing N and Sun X: Cardioprotective effects of Notoginsenoside R1 against ischemia/reperfusion injuries by regulating oxidative stress- and endoplasmic reticulum stress- related signaling pathways. Sci Rep. 6:217302016. View Article : Google Scholar : PubMed/NCBI

37 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

38 

Liao B, Dong S, Xu Z, Gao F, Zhang S and Liang R: LncRNA Kcnq1ot1 renders cardiomyocytes apoptosis in acute myocardial infarction model by up-regulating Tead1. Life Sci. 256:1178112020. View Article : Google Scholar : PubMed/NCBI

39 

Lai L, Liu Y, Liu Y, Zhang N, Cao S, Zhang X and Wu D: Role of endoplasmic reticulum oxidase 1α in H9C2 cardiomyocytes following hypoxia/reoxygenation injury. Mol Med Rep. 22:1420–1428. 2020. View Article : Google Scholar : PubMed/NCBI

40 

Walter P and Ron D: The unfolded protein response: From stress pathway to homeostatic regulation. Science. 334:1081–1086. 2011. View Article : Google Scholar : PubMed/NCBI

41 

Oyadomari S and Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11:381–389. 2004. View Article : Google Scholar : PubMed/NCBI

42 

Durante W: Targeting endoplasmic reticulum stress in hypoxia-induced cardiac injury. Vascul Pharmacol. 83:1–3. 2016. View Article : Google Scholar : PubMed/NCBI

43 

Levy JMM, Towers CG and Thorburn A: Targeting autophagy in cancer. Nat Rev Cancer. 17:528–542. 2017. View Article : Google Scholar : PubMed/NCBI

44 

Lin R, Su Z, Tan X, Su Y, Chen Y, Shu X, Liang S, Wang J and Xie S: Effect of endoplasmic reticulum stress and autophagy in the regulation of post-infarct cardiac eepair. Arch Med Res. 49:576–582. 2018. View Article : Google Scholar : PubMed/NCBI

45 

Wiersma M, Meijering RAM, Qi XY, Zhang D, Liu T, Hoogstra-Berends F, Sibon OCM, Henning RH, Nattel S and Brundel BJJM: Endoplasmic reticulum stress is associated with autophagy and cardiomyocyte remodeling in experimental and human atrial fibrillation. J Am Heart Assoc. 6:e0064582017.doi: 10.1161/JAHA.117.006458. View Article : Google Scholar : PubMed/NCBI

46 

Holdt LM and Teupser D: Long noncoding RNA ANRIL: Lnc-ing genetic variation at the chromosome 9p21 locus to molecular mechanisms of atherosclerosis. Front Cardiovasc Med. 5:1452018. View Article : Google Scholar : PubMed/NCBI

47 

Chi JS, Li JZ, Jia JJ, Zhang T, Liu XM and Yi L: Long non-coding RNA ANRIL in gene regulation and its duality in atherosclerosis. J Huazhong Univ Sci Technolog Med Sci. 37:816–822. 2017.doi: 10.1007/s11596-017-1812-y. PubMed/NCBI

48 

Chang JC, Hu WF, Lee WS, Lin JH, Ting PC, Chang HR, Shieh KR, Chen TI and Yang KT: Intermittent hypoxia induces autophagy to protect cardiomyocytes from endoplasmic reticulum stress and apoptosis. Front Physiol. 10:9952019. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

February-2021
Volume 23 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Li J, Xie J, Wang Y, Gan Y, Wei L, Ding G, Ding Y and Xie D: Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324. Mol Med Rep 23: 116, 2021
APA
Li, J., Xie, J., Wang, Y., Gan, Y., Wei, L., Ding, G. ... Xie, D. (2021). Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324. Molecular Medicine Reports, 23, 116. https://doi.org/10.3892/mmr.2020.11755
MLA
Li, J., Xie, J., Wang, Y., Gan, Y., Wei, L., Ding, G., Ding, Y., Xie, D."Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324". Molecular Medicine Reports 23.2 (2021): 116.
Chicago
Li, J., Xie, J., Wang, Y., Gan, Y., Wei, L., Ding, G., Ding, Y., Xie, D."Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324". Molecular Medicine Reports 23, no. 2 (2021): 116. https://doi.org/10.3892/mmr.2020.11755