LncRNA DLX6-AS1 promotes necroptosis through PI3K/AKT/mTOR signaling pathway in myocardial ischemia/reperfusion injury mice

Abstract

Myocardial ischemia/reperfusion injury (MIRI) leads to life-threatening myocardial infarction. Necroptosis is involved in MIRI. The lncRNA DLX6-AS1 was found to participate in the context of MIRI. In this article, we examined the effect of DLX6-AS1 on ischemia perfusion-induced myocardial injury caused by necroptosis and the underlying potential mechanism. We constructed a mouse model of MIRI, and hematoxylin-eosin staining, TUNEL staining, echocardiology, Western blotting (WB), and reverse transcriptase-polymerase chain reaction (RT‒PCR) were employed to evaluate myocardial infarction and necroptosis. Rat H9c2 cells were induced by hypoxia-reoxygenation (H/R), and we analyzed the possible mechanism by overexpressing and inhibiting DLX6-AS1 expression. Cell Counting Kit 8, lactate dehydrogenase (LDH), flow cytometry, RT‒PCR, and WB were utilized to examine cell injury, apoptosis, and relative protein expression. MIRI induced myocardial damage and impaired cardiac function in mice. Ischemia/reperfusion injury results in necroptosis, a process that Nec-1 inhibits. The mRNA levels of RIP1, RIP3, and MLKL were increased, and DLX6-AS1 demonstrated an increase in the IR model, indicating that DLX6-AS1 was associated with necroptosis. Furthermore, H/R-induced H9C2 cells exhibited high LDH levels, low cell activity, and severe apoptosis levels. DLX6-AS1 overexpression caused cell damage and necroptosis and increased the expression of proteins, including PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR. DLX6-AS1 inhibition suppressed necroptosis and cell damage through the PI3K/AKT/mTOR signaling pathway. Myocardial ischemia‒reperfusion injury induces necroptosis, and DLX6-AS1 promotes this process through the PI3K/AKT/mTOR signaling pathway.

Introduction

Myocardial infarction (MI)^1–3 has a high morbidity and mortality rate globally, and it is a severe and life-threatening disease in the world⁴. Globally, the number of cardiovascular diseases deaths was 18.6 million in 2019, increased significantly than 1990^5,6. Our country has the similar high mortality rates⁷. MI is becoming increasingly prevalent among younger patients; thus, many consider it a public health concern⁸.

Prompt reperfusion of the coronary artery related to infarction is the most effective treatment option for MI. However, reperfusion can produce a series of functional disorders and structural damage to the heart muscles in the ischemic myocardium, including myocardium stunning, no reflow phenomenon, reperfusion arrhythmia, and myocardial cell death⁹. Conversely, the precise mechanism of MIRI remains unknown, and there currently exists no efficacious treatment for this injury.

Necroptosis, a type of programmed necrosis, differs from apoptosis. The pathological characteristics of necroptosis include tissue swelling, cell plasma membrane rupture, and an acute inflammatory reaction caused by the release of many endogenous damage-related molecules¹⁰. Necroptosis is primarily induced by receptor-interacting protein kinase 1 (RIP1/RIPK1), receptor-interacting protein kinase 3 (RIP3/RIPK3), and downstream mixed lineage kinase domain-like protein (MLKL). RIP3 is activated by RIP1 when TNFR1 initiates necroptosis, independent of caspase-8. After activation, RIP3 catalyzes MLKL phosphorylation, and the RIP1-RIP3-MLKL complex promotes the formation of MLKL oligopolies (mostly tripoles or quadrupoles), which are transported to the cell plasma membrane. Furthermore, this process destroys the membrane, leading to cell dissolution and death¹¹. Necroptosis is involved in many cardiovascular diseases, including atherosclerosis, MI, and abdominal atrophy¹². A previous study reported that myocardial injury is considerably induced by necroptosis¹³. Based on the impact of necroptosis in MIRI, in addition to the traditional pathway in the formation of necrosis bodies, RIPK3 is directly involved in necroptosis through the downstream axis -Ca^{2 +} calmodulin-dependent protein kinase II (CaMK II)¹⁴, independent of RIPK1, to promote Met281/282 and Thr287 phosphorylation oxidation. Furthermore, necroptosis occurs when the concentration of calcium ions in the cell increases and the mitochondrial conversion pore opens¹⁵. Nec-1 is a RIPK1 inhibitor that prevents necroptosis and relieves myocardial injury. A previous study reported that Nec-1 may shrink the area of infarction and improve cardiac function after infarction in ischemia/reperfusion (IR) models of mice¹⁶. Furthermore, other previous studies reported close interactions between MIRI and necroptosis.

Long noncoding RNAs (lncRNAs) are RNAs with > 200 nt and few protein-encoding functions^17,18. However, it regulates the expression of coding genes^19,20. Different biological processes rely on lncRNAs through various mechanisms^21,22. Previous studies reported that lncRNA is involved in necroptosis; the Tran D study reported that lncRNA inc00176 regulates gene expression that affects liver cancer cell necroptosis²³. Another study reported that lncRNA necrosis-related factor induces myocardial necroptosis by selectively modulating miR-873 and regulating the activity of RIPK1/RIPK3²⁴. We initially identified lncRNA DLX6-AS1 as an enhancer without remote homeoframe 5/6 transferred activity, and it is specifically expressed in cells, tissues, or tumors. It can participate in various regulatory mechanisms of physiological processes²⁵. DLX6-AS1 upregulates and promotes the malignant transformation of cancers of the colon, stomach, and liver²⁶. Additionally, the suppression of DLX6-AS1 expression has been shown to impede cell migration, proliferation and invasion in tumor cells, suggesting an inherent anti-neoplastic property of DLX6-AS1^27,28. Past articles have confirmed that DLX6-AS1 is more highly expressed in the human brain. Hu²⁹ reported that DLX6-AS1 engages in the neural apoptosis process resulting from cerebral ischemia/reperfusion injury as a ceRNA via the DLX1-AS1/miR-149-3p/BOK axis. Wang et al.³⁰ stated that DLX6-AS1 contributed to MIRI through the DLX6-AS1/miR-204-5p/FBXW7 axis, suggesting that DLX6-AS1 could emerge as a viable therapeutic target for MIRI.

A study proved that suppressing the PI3K/AKT/mTOR pathway could promote autophagy and inhibit cell migration and cell cycle arrest in gastric cancer cells³¹. Another previous study documented that DLX6-AS1 facilitates cell growth, invasion, and colon cancer cell migration through the PI3K/AKT/mTOR pathway and suppresses apoptosis in patients with colon cancer²⁶. Therefore, we hypothesize that DLX 6-AS1 may affect the process of necroptosis in the myocardium through the PI3K/AKT/mTOR pathways in MIRI. Here, we constructed IR mouse models, explored the action of DLX6-AS1 in MIRI and investigated the potential mechanism involving the PI3K/AKT/mTOR pathway in vitro.

Materials and methods

IR injury mouse model

SPF male C57BL/6 mice aged between six and eight weeks were purchased from the Animal Experiment Center of Tianjin Medical University. The research adhered to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and received approval from the Animal Review Board of Cangzhou Central Hospital.

The mice underwent anesthesia via intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg. On the left side between the third and fourth intercostal spaces, thoracotomy was performed. The left anterior descending coronary artery of the mouse was tied using a 6 − 0 silk suture. ST-segment elevation and a high or reverse T wave on electrocardiograph were utilized to accurately diagnose ischemia. After 30 min of ligation, the suture was released, and blood flow was restored for 120 min. The identical procedure was executed in the sham group; however, the silk suture was untied. Mice in different groups were injected with 1.65 mg/kg Nec-1 (MCE, Shanghai, China, CAS No. 4311-88-0)³² or vehicle (0.05% DMSO) intraperitoneally for 15 min before the operation. Four groups (n = 10 per group) were divided, including sham with vehicle, sham with Nec-1, IRI with vehicle, and IRI with NEC-1. These mice were sacrificed 24 h after the operation.

Animal anesthesia and euthanasia

Mice were anesthetized with isoflurane using a calibrated vaporizer (induction at 3–5% isoflurane in oxygen until loss of the righting reflex, followed by maintenance at 1–2% as required for procedures). Euthanasia was performed by carbon dioxide inhalation in a chamber initially containing room air, with 100% CO₂ introduced at a controlled gradual displacement rate of 20–30% of the chamber volume per minute until respiratory arrest. Death was confirmed by the absence of respiration and heartbeat according to institutional animal care guidelines.

Echocardiography evaluation

We employed M-mode echocardiography (Vevo 2100, Canada) to examine left ventricular systolic function 24 h after reperfusion. The mice underwent anesthesia via intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg, and hair removal cream was utilized to remove hairs on their chests. The anterior chest walls were completely exposed, and the mice were placed supine on the ultrasound table. The extent of the left ventricular papillary muscle was marked on the parasternal short axis section. The left ventricular short axis shortening (FS) and ejection fraction (EF) were measured via M-mode ultrasonography.

Histological analysis

When echocardiography was finished, the mice were sacrificed with deep anesthesia, and their hearts were quickly harvested. Myocardial tissues were obtained, placed in 4% paraformaldehyde, and subsequently fixed for 24 h. The specimens underwent dehydration, paraffin embedding, and subsequent sectioning into 4 μm-thick slices. Hematoxylin-eosin (H&E) staining was performed on the paraffin-embedded sections. H&E staining kits were acquired from Beyotime, Shanghai, China (Cat: C0105M). The pathological alterations in myocardial tissue were observed and captured using an optical microscope. The scoring standard includes three fields that were randomly selected under a 40-fold objective lens to view the entire left ventricular myocardium section. The distribution of cardiomyopathy was observed from four aspects of cardiomyopathy: necrosis, hemorrhage, intrastromal edema, and neutrophil infiltration, and scores were calculated. The ratio of visual fields for each lesion (the number of visual fields with cardiomyopathy/the total number of visual fields observed under the microscope) was calculated. Focal lesions with a proportion of the lesion field ≤ 1/4 were scored as 1; those with a proportion of the lesion field between 1/4 and ½ were scored as 2; diffuse lesions with a proportion of the lesion field ≥ 1/2 were scored as 3. Employing the TUNEL Apoptosis Assay Kit (Proteintech, China, Cat: PF00006), terminal deoxynucleotidyl transferase (TdT) was utilized to enzymatically incorporate deoxyribonucleotide-biotin (FITC) derivatives onto the 3’-ends of DNA strands, thus allowing for the fluorescent detection of apoptotic cells³³.

Quantitative real-time PCR (qRT‒PCR)

Myocardium and H9C2 cells were treated with TRIzol reagent (Invitrogen, USA, Cat: 15596026) to procure total RNA, followed by the utilization of PrimeScript RT Master Mix (Takara, Tokyo, Japan, Cat: RR036Q) for the synthesis of complementary DNA from the obtained total RNA. Assessment of the relative expression level of mRNAs was performed using Terra qPCR Direct TB Green Premix (Takara, Tokyo, Japan, Cat: 638319). Normalization of all relative lncRNA and mRNA expression levels was conducted with respect to GAPDH. Quantification of the results was carried out utilizing the 2–ΔΔCT method for analysis³⁴. Table 1 illustrates each primer sequence.

Table 1.

Primer sequences.

Gene name	Primer sequences (5`-3`)
DLX6-AS1	Forward primer 5`-GACTGACTGAGCGTGGAAGTTGC-3` Reverse primer 5`-TCTGGGCGTAGGTTTCTCTCTGG-3`
RIP1	Forward primer 5`- TTCGCTGGTGATGGAGTA − 3` Reverse primer 5`- AGGATATTCTCAGGCTTCAG-3`
RIP3	Forward primer 5`- GGCTCTCGTCTTCAACAA − 3` Reverse primer 5`- ACTGTGCTTGGTCATACTT-3`
MLKL	Forward primer 5`- TTGAAGGCTGTGATTCTAAG-3` Reverse primer 5`- CTCCAAGATTCGTTCTCTG-3`
GAPDH	Forward primer 5`-GTCAGCCGCATCTTCTTTTG-3` Reverse primer 5`-GCGCCCAATACGACCAAATC-3`

Western blotting (WB)

Radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China, Cat: R0046) was added to myocardial tissues or H9C2 cells, the concentration of all proteins was determined using the bicinchoninic acid protein assay (BCA) method, and the kit was purchased from Beyotime, Shanghai, China (Cat: P0009). Protein samples from each group were separated on SDS‒PAGE gels (Beyotime, Shanghai, China, Cat: P0012A) and, through electrophoresis, transferred to PVDF membranes (Beyotime, Shanghai, China, Cat: FFP24). The PVDF membranes were incubated with primary antibodies, RIP1 (Cat: ab300617, 1:1000 dilution), RIP3 (Cat: ab255705, 1:500 dilution), p-MLKL (Cat: ab196436, 1:1000 dilution), MLKL (Cat: ab184718, 1:1000 dilution), GAPDH (Cat: ab181602, 1:1000 dilution), PI3K (Cat: ab191606, 1:1000 dilution), p-PI3K (Cat: ab74136, 1:1000 dilution), AKT (Cat: ab179463, 1:1000 dilution), p-AKT (Cat: ab38449, 1:1000 dilution), mTOR (Cat: ab134903, 1:1000 dilution), and p-mTOR (Cat: ab314037, 1:1000 dilution). Membranes were incubated at 4 °C overnight and rinsed thrice with TBST. The secondary antibody, HRP goat anti-rabbit IgG (Cat: ab6721, 1:2000 dilution), was incubated at 37 °C for 1 h. All of the antibodies mentioned above were acquired from Abcam (Cambridge, MA, USA). ECL chemiluminescence (Beyotime, Shanghai, China, Cat: P0018S) was utilized to visualize protein bands, and Image-Pro Plus 6.0 was employed for grayscale analysis.

Cell culture

The rat embryonic cardiomyocyte cell line H9C2 was purchased from the Chinese Academy of Sciences (CAS) Cell Bank. H9C2 cells were cultured in DMEM (Cat: C0891) containing 10% fetal bovine serum (FBS, Cat: C0226) at 37 °C with 5% CO₂. The reagents above were all purchased from Beyotime, Shanghai, China. Passaging was performed at 1:3 when the cell density was 80%–90%. H9C2 cells were counted using a blood cell counter at 1 × 10⁵ cells/mL density, and the experiment commenced by seeding cells into 96-well plates at a volume of 100 µL per well, followed by an incubation period at 37 °C for 12 h. Upon cellular adherence to the substrate, the supernatant was subsequently removed. Then, 100 µL of serum-free and sugar-free DMEM culture medium was added and cultured in an anaerobic incubator (1% O₂, 5% CO₂, and 94% N₂) for 4 h, and the supernatant was removed. Serum-containing DMEM culture medium was added and incubated in a normal environment (5% CO₂, 95% O₂, 37 °C) for 3 h to induce the H/R model³⁵. Serum-containing DMEM culture medium was employed as a blank control, and five replicate wells were arranged for each concentration. The laboratory group received 10 mmol/L NEC-1. The PI3K inhibitor LY294002 (Sigma‒Aldrich, St. Louis, MO, USA, Cat: L9008) was added 1 h before H/R at a concentration of 25 µmol/L and then incubated in the laboratory group for 48 h^36,37.

Cell transduction

H9C2 cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. These cells were all saved in a CO₂ incubator at 37 °C and 5% CO₂. When the cell confluence reached 70%–80%, H9C2 cells were transfected by using Lipofectamine 2000 Transfection Reagent (Invitrogen, USA, Cat: 11668500). The DLX6-AS1 overexpression model was constructed using a pcDNA3.1 vector inserted into DLX6-AS1 (pc-DLX6-AS1). The DLX6-AS1 knockdown model was constructed by targeting DLX6-AS1 with small interfering RNA (si-DLX6-AS1). After 24 h of transfection, fluorescence changes were observed using fluorescence microscopy, and the expression level of DLX6-AS1 was assessed through qRT‒PCR to confirm the transfection outcomes.

Cell counting kit 8 (CCK8) assay

Cardiomyocyte viability was detected by the CCK-8 cell viability assay kit (Beyotime, Shanghai, China, Cat: C0041). H9C2 cardiomyocytes were inoculated into 96-well plates, and after the cells were attached to the wall, the cells were treated according to the instructions of each group. After treatment, 100 µL of a 10% CCK-8 solution was introduced to the cells in each well, followed by an incubation period at 37 °C for 2 h. The optical density was measured at 450 nm using a BioTek enzyme labeling instrument. Cell survival rate (%) = cell optical density value of each group/cell optical density value of LG-Control group × 100%.

Lactate dehydrogenase (LDH) test

After modeling, the supernatants of different groups of cells were collected, and the LDH kit, which was purchased from Beyotime, Shanghai, China (Cat: C0016), was utilized to calculate the LDH levels in each group according to the manufacturer’s instructions.

Flow cytometry

An Annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China, Cat: C1062S) was utilized to determine the apoptotic ratio of H9C2 cells in different groups, and a mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China, Cat: C2003S) was also used. A BD FACSCalibur (BD Bioscience, San Jose, USA) was utilized to test the relative index.

Statistical analysis

SPSS 26.0 statistical software was used to analyze all the data. The data are presented as the mean ± standard deviation (x ± s). Student’s t test was utilized for two-way comparisons between two different groups, and one-way ANOVA was utilized for comparisons between multiple groups. Pearson’s and Spearman’s correlation were used to detect the relation between two groups. A significance level of p < 0.05 was considered statistically significant. The method in this section is mainly based on previous studies with minor revisions^38,39.

Results

DLX6-AS1 was significantly elevated after MIRI and correlated with necroptosis factors, including RIP1, RIP3, and p-MLKL

We constructed a mouse IRI model to examine whether necroptosis occurs during myocardial ischemia/reperfusion injury. Based on the HE staining of the myocardium tissue in the IRI and sham groups (Fig. 1A), the IRI group had significantly more severe damage in their myocardial tissues than the mice in the sham group (p < 0.05), and our scoring system confirmed this finding. Based on the myocardial TUNEL staining and scoring between the two groups of mice (Fig. 1C), the IRI group had a significantly higher degree of apoptotic cells and an increased rate of apoptosis than the sham group (p < 0.05). Additionally, cardiac ultrasound examination of the mice in the two groups showed that the levels of FS and EF in the mice in the IRI group were lower than those in the sham group (Fig. 1E). Furthermore, the heart rates of mice in the IRI group were significantly increased compared with those in the sham group (p < 0.05), suggesting that MIRI in mice substantially impairs cardiac function.

Fig. 1.

MIRI in mice was related to programmed necrosis. DLX6-AS1 expression was significantly increased in the myocardium after MIRI. (A-B) Pathological changes in the myocardium were detected by HE staining. (C-D) Apoptotic levels in the myocardium were detected by TUNEL staining. (E-F) The cardiac function test by cardiac ultrasound for FS%, EF% and heart rate. (G-H) The protein levels of RIP1, RIP3, p-MLKL and MLKL in myocardial tissues were determined by WB. Magnification ×400; ****p < 0.0001 vs. sham; ***p < 0.001 vs. sham. Scale bar = 100 μm.

The myocardial tissues of mice in both groups were subjected to WB to measure the levels of necroptosis-related indexes RIP1, RIP3, and p-MLKL. We found that the levels of RIP1, RIP3, and p-MLKL in mice in the IRI group were elevated significantly compared with those in the sham group (Fig. 1G), reflecting the incidence of necroptosis in MIRI.

Based on the expression level of lncRNA DLX6-AS1 in the myocardial tissues of the two groups of mice (Fig. 2A), the level of DLX6-AS1 in the mice of the IRI group was significantly increased compared with that of mice in the sham group (p < 0.05), indicating that DLX6-AS1 may be related to myocardial ischemia/reperfusion injury. Based on the correlation analysis between DLX6-AS1 and RIP1, RIP3, and MLKL (Fig. 2B-D), DLX6-AS1 was significantly correlated with RIP1, RIP3, and MLKL.

Fig. 2.

Correlation analysis between DLX6-AS1 and RIP1, RIP3 and MLKL was performed by Pearson and Spearman correlation analysis. (A) The expression of DLX6-AS1 in two groups. (B) The level of DLX6-AS1 was positively related to RIP1. (C) The expression of DLX6-AS1 was positively related to RIP3. (D) The expression of DLX6-AS1 was positively related to MLKL.****p <0.0001 vs sham. 

NEC-1 attenuated myocardial injury and improved cardiac function in MIRI mice by inhibiting RIP1, affecting DLX6-AS1 expression levels in the mouse myocardium

Based on the HE staining of the myocardium in the four groups of mice (Fig. 3A-B), together with the scoring system, we noticed a significant difference (p < 0.05) among the four groups. The scoring of HE-stained slices showed that the myocardium of mice in the IRI group was more severely damaged than that of mice in the sham group. However, in the two IRI model groups, the myocardia of mice in the Nec-1 intervention group were more improved than those in the nonintervention group (p < 0.05).

Fig. 3.

NEC-1 attenuated myocardial injury and improved cardiac function in MIRI mice by inhibiting RIP1 and affecting DLX6-AS1 expression levels in mouse myocardium. (A-B) Pathological changes in the myocardium were detected by HE staining. Scale bar = 100 μm. (C-D) Apoptotic levels in the myocardium were detected by TUNEL staining. Scale bar = 100 μm. (E-F) The cardiac function test by cardiac ultrasound for FS, EF and heart rate. (G-H) The protein levels of RIP1, RIP3, p-MLKL and MLKL in the myocardium were determined by WB analysis. (I) DLX6-AS1 relative expression by qRT‒PCR. (J-K) Relative expression of downstream factors, including PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR, as determined by WB. Magnification×400; **** p < 0.0001 vs. sham + vehicle; *** p < 0.001 vs. sham + vehicle; #### p < 0.0001 vs. IRI + vehicle; ### p < 0.001 vs. IRI + vehicle; # p < 0.05 vs. IRI + vehicle.

Figure 3C-D depicts the TUNEL staining results of the myocardial tissues of the four groups of mice. We found a higher rate of apoptosis in the IRI group than in the sham group (p < 0.05). However, apoptotic cells were reduced in the myocardium of IRI mice after Nec-1 intervention, and a significant decrease in the apoptotic rate was recommended (p < 0.05).

The cardiac function examination findings in the four groups of mice showed that the FS and EF of mice in the IRI group were higher than those of mice in the sham group, p < 0.05. The heart rates of mice in the above groups showed opposite results. However, the FS and EF of mice showed decreases in the Nec-1 intervention groups compared with the vehicle intervention groups, and the heart rates showed the opposite trend. All results above demonstrated that cardiac function decreased in mice in the IRI group; however, there was an improvement in the mice in the Nec-1 intervention group (p < 0.05) (Fig. 3E-F).

Furthermore, WB assays were performed on myocardial tissues from the four groups of mice to detect RIP1, RIP3, MLKL and p-MLKL expression levels in the myocardium, and the level of RIP1 was significantly decreased in the Nec-1 intervention group under IRI modeling (p < 0.05). Additionally, RIP3, MLKL and p-MLKL levels in the Nec-1 intervention group were also decreased in the same groups (p < 0.05). Furthermore, the levels of RIP1, RIP3, MLKL and p-MLKL in the IRI group were obviously elevated (p < 0.05) (Fig. 3G-H).

Figure 3 depicts lncRNA DLX6-AS1 levels in the four different groups. We found that the expression of DLX6-AS1 (Fig. 3I) was significantly elevated among the mice in the IRI group compared with the sham group, but we found that the decrease in DLX6-AS1 expression after Nec-1 treatment was not statistically significant (p > 0.05). However, a certain decreasing tendency was observed after inhibition by Nec-1. We applied WB technology to detect the expression of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR in the four groups (Fig. 3J-K) and found that the three downstream factors and their phosphorylated forms were elevated to some degree in the IRI group and that these indexes were decreased after Nec-1 intervention (p < 0.05).

Necroptosis indicators were elevated in H9C2 cells with hypoxia/reoxygenation injury, and Nec-1 attenuated the degree of injury

We utilized rat H9C2 cells to trigger hypoxia/reoxygenation (H/R) and intervened by administering Nec-1. We found that the CCK8 level of H9C2 cells in the H/R group was decreased, indicating a decrease in cell viability, whereas the CCK8 activity of H9 C2 cells was increased after Nec-1 intervention. However, it was lower than that of the sham group and was higher than that of the disease group (p < 0.05) (Fig. 4A). We performed LDH assays on the four groups and found that LDH levels in hypoxia/reoxygenation-induced H9C2 cells were elevated, which indicated that the cells were damaged (Fig. 4B). A flow cytometry assay was performed on the four groups, and we observed substantial apoptosis in hypoxia/reoxygenation-induced H9C2 cells, which could be ameliorated by Nec-1 (Fig. 4C-D). Figure 4E-F displays the RIP1, RIP3, MLKL and p-MLKL expression levels in the four groups. Necroptosis-related indicators (RIP1, RIP3, MLKL and p-MLKL) were differentially elevated in hypoxia/reoxygenation-induced H9C2 cells in the H/R group (p < 0.05). The expression levels of DLX6-AS1 in the four groups were further detected, and the outcomes (Fig. 4G) showed that the DLX6-AS1 level was elevated in the H/R group (p < 0.05). The downstream factors PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR were detected in the four groups of cells, and we found that the above three factors and their phosphorylated forms were differentially elevated in the H/R group, and the difference in expression was statistically significant (Fig. 4H-I).

Fig. 4.

Hypoxia/reoxygenation-induced H9c2 cells showed necroptosis, and DLX6-AS1 could affect necroptosis through the PI3K/AKT/mTOR signaling pathway. (A) Cell viability tested by CCK8. (B) Cell injury was tested by an LDH assay kit. (C-D) Flow cytometry was utilized to test the apoptosis of H9c2 cells. (E-F) RIP1, RIP3, MLKL and p-MLKL protein expression was tested by WB. (G) DLX6-AS1 expression by qRT‒PCR. (H-I) PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR protein expression was tested by WB. ****p < 0.0001 vs. sham + vehicle; ####p < 0.0001 vs. H/R + vehicle; ##p < 0.01 vs. H/R + vehicle; #p < 0.05 vs. H/R + vehicle.

DLX6-AS1 overexpression accentuated necroptosis in H9C2 cells through the PI3K/AKT/mTOR pathway

We tested CCK8 viability using DLX6-AS1 overexpression through cell transfection (Fig. 5A) and observed that the viability of H9C2 cells in the H/R group was significantly decreased, and DLX6-AS1 overexpression further decreased the viability of H9C2 cells (p < 0.05).

Fig. 5.

Overexpression of DLX6-AS1 promoted H9c2 cell necroptosis and injury. (A) Cell viability tested by CCK8. (B) Cell injury was tested by an LDH assay kit. (C-D) Flow cytometry was utilized to test the apoptosis of H9c2 cells. (E-F) RIP1, RIP3, p-MLKL and MLKL protein levels were tested by WB. (G) DLX6-AS1 expression by qRT‒PCR. (H-I) PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR protein expression was tested by WB. (J-K) RIP1, RIP3, p-MLKL and MLKL protein levels were tested by WB. (L-M) PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR protein expression was tested by WB. ****p < 0.0001 vs. sham + NC; **p < 0.01 vs. sham + NC; *p < 0.05 vs. sham + NC; ####p < 0.0001 vs. H/R + NC; ###p < 0.001 vs. H/R + NC; ##P < 0.01 vs. H/R + NC; #P < 0.05 vs. H/R + NC.

We detected LDH levels (Fig. 5B) and observed that necrosis of H9C2 cells could be exacerbated by overexpression of DLX6-AS1 (p < 0.05).

Additionally, we detected H9C2 cells by flow cytometry (Fig. 5C-D) and found that the number of apoptotic H9C2 cells after DLX6-AS1 overexpression was significantly increased, indicating that DLX6-AS1 might promote apoptosis in H9C2 cells.

The expression of RIP1, RIP3, MLKL and p-MLKL was detected in H9C2 cells (Fig. 5E-F), and we observed that after transfecting DLX6-AS1 plasmids, the levels of RIP1, RIP3, MLKL and p-MLKL in the H/R group were elevated, indicating that DLX6-AS1 may induce the development of necroptosis in H9C2 cells.

We detected DLX6-AS1 levels in the four groups (Fig. 5G) and found that DLX6-AS1 levels in H9C2 cells treated with hypoxia/reoxygenation were significantly elevated (p < 0.05). However, DLX6-AS1 levels were all elevated in the transfected cells, indicating successful transfection.

By detecting the levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR (Fig. 5H-I), we observed that H9C2 cells in the H/R group exhibited a notable trend of elevation in the levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR subsequent to the overexpression of DLX6-AS1 (p < 0.05). However, after PI3K was inhibited, the expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR were decreased (p < 0.05), and the same changes were found (Fig. 5J-K) in the levels of RIP1, RIP3, MLKL and p-MLKL (p < 0.05). After the PI3K inhibitor LY294002 was applied in these groups, the expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR were decreased (Fig. 5L-M), and the expression levels of RIP1, RIP3, MLKL and p-MLKL were decreased in the same way. This result indicated that DLX6-AS1 might influence the necroptosis process of H9C2 cells through the PI3K/AKT/mTOR pathway.

DLX6-AS1 deficiency alleviated necroptosis in H9C2 cells

We constructed DLX6-AS1 knockdown plasmids and detected CCK8 activity in H9C2 cells in different groups. The outcomes (Fig. 6A) show that after the knockdown of DLX6-AS1, the viability of H9C2 cells in the H/R group was increased; however, it was lower than that of the sham group. The LDH assay data (Fig. 6B) similarly revealed that after the knockdown of DLX6-AS1, the damage to H9C2 cells in the sham group was significantly decreased compared with that in the H/R group (p < 0.05). As detected by flow cytometry (Fig. 6C-D), we found that the rate of apoptotic cells in the H/R group after DLX6-AS1 knockdown was higher than that of the H/R group alone. WB technology further detected the levels of RIP1, RIP3, MLKL and p-MLKL proteins in the cells of each group (Fig. 6E-F), and the outcomes showed that the indicators of necroptosis were decreased after the knockdown of DLX6-AS1. Furthermore, after examining the DLX6-AS1 levels in four groups of H9C2 cells (Fig. 6G), we identified that DLX6-AS1 levels in cells transfected with the si-DLX6-AS1 plasmid were significantly decreased, suggesting the success of transfection. We examined the levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR proteins in the four groups of cell models (Fig. 6H-I). The results illustrated that the levels of the above three proteins and their phosphorylated forms showed a downward trend in the H/R cell model with DLX6-AS1 knockout, which reinforced our speculation that DLX6-AS1 affects necroptosis through the PI3K/AKT/mTOR pathway. Together, these results suggest that the specific regulatory mechanism by which lncRNA DLX6-AS1 promotes necroptosis in myocardial ischemia/reperfusion injury by activating the PI3K/AKT/mTOR signaling pathway (Fig. 7).

Fig. 6.

Silencing DLX6-AS1 inhibited H9c2 cell necroptosis and injury. (A) Cell viability tested by CCK8. (B) Cell injury was tested by an LDH assay kit. (C-D) Flow cytometry was utilized to test the apoptosis of H9c2 cells. (E-F) RIP1, RIP3 p-MLKL and MLKL protein expression was tested by WB. (G) DLX6-AS1 expression by qRT‒PCR. (H-I) PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR protein expression was tested by WB. ****p < 0.0001 vs. sham + NC; ***P < 0.001 vs. sham + NC; **p < 0.01 vs. sham + NC; ####p < 0.0001 vs. H/R + NC; ###p < 0.001 vs. H/R + NC; ##P < 0.01 vs. H/R + NC.

Fig. 7.

A schematic diagram representing the mechanism of this study. Figure created in BioRender. SU, Y. (2026) https://BioRender.com/p43o3zq.

Discussion

MI is widely recognized as one of the most severe and life-threatening diseases. With the advancement of diagnostic and therapeutic methods, myocardial ischemia caused by reperfusion-induced injury is gradually gaining attention. MIRI is a complex process involving multiple molecular mechanisms⁴⁰. Past papers have reported that many lncRNAs are important in the pathophysiologic progression of MIRI. Both in vivo and in vitro animal experiments and clinical studies have revealed different degrees of up- or downregulation of lncRNAs in response to MIRI, suggesting that lncRNAs may serve as markers of MIRI in patients with MI and even as targets for therapy⁴¹.

Necroptosis plays a role in MIRI through two mechanisms: the formation of necrotic vesicle complexes and RIPK3, which directly causes necroptosis of cells through calcium-dependent protein kinases. Our results show that necroptosis is present in a mouse model of IRI and can be ameliorated by the RIP1 inhibitor Nec-1. Here, we sought to explore whether DLX6-AS1 modulates necroptosis via the RIPK1-dependent pathway. Nec-1, a well-characterized RIPK1 inhibitor, was utilized to selectively abrogate RIPK1 activity. Comparative analysis of necroptotic markers with and without Nec-1 intervention enabled us to ascertain the contribution of RIPK1 to DLX6-AS1-mediated necroptosis. This experimental strategy establishes a mechanistic connection between DLX6-AS1 and RIPK1-dependent necroptotic signaling cascades. MLKL phosphorylation represents the final execution step of necroptosis. In our study, the increase in p-MLKL levels and its effective suppression by Nec-1 provide molecular evidence distinguishing necroptosis from apoptosis⁴².

Previous studies reported that DLX6-AS1 plays a crucial role in the progression of many tumors, and some studies found that it induces apoptosis of neuronal cells in cerebral ischemia/reperfusion. In acute kidney injury, DLX6-AS1 mediates lipopolysaccharide-induced toxic cellular pyroptosis in renal tubular epithelial cells²⁵. However, in MIRI, the effect of the DLX6-AS1/miR-204-5p/FBXW7 axis increased cardiomyocyte injury. We revealed that DLX6-AS1 levels were substantially elevated in cardiomyocytes of IRI model mice by comparing the IRI model with the sham model, suggesting an elevation of DLX6-AS1 levels after myocardial ischemia/reperfusion injury. Correlation analysis showed that DLX6-AS1 was significantly correlated with RIP1 mRNA, an indicator of necroptosis, and positively correlated with RIP3 and MLKL levels, which suggests that DLX6-AS1 influences necroptosis in the myocardium of mice with MIRI. Necrostatin-1 treatment markedly suppressed MLKL phosphorylation, further confirming that the observed cell death was RIPK1-dependent necroptosis. We found that there was no statistical significance between the two groups after Nec-1 treatment. We believe that Nec-1 does not exert its cardioprotective effect by directly regulating DLX6-AS1 expression but rather through its well-established role as a specific RIPK1 kinase inhibitor, thereby blocking the necroptosis pathway. Nec-1 does not exert its cardioprotective effect by directly regulating DLX6-AS1 expression but rather through its well-established role as a specific RIPK1 kinase inhibitor, thereby blocking the necroptosis pathway. Furthermore, we found that DLX6-AS1 might affect necroptosis of the myocardium through the PI3K/AKT/mTOR pathway by detecting the levels of the downstream molecules PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR.

We subjected H9C2 cells to hypoxia/reoxygenation. Further study revealed that the viability of cells in the H/R group was significantly reduced; however, the LDH level, apoptosis level, necroptosis factor, and DLX6-AS1 expression were significantly elevated. However, all of the above manifestations could be inhibited by Nec-1. Our results show that DLX6-AS1 overexpression promotes in vitro necroptosis, as evidenced by increased p-MLKL levels. Nec-1-mediated RIPK1 inhibition partially attenuated this effect, indicating that DLX6-AS1-induced necroptosis is at least partially RIPK1 dependent. Additionally, PI3K inhibitor treatment partially reversed necroptosis, suggesting that the PI3K/AKT/mTOR pathway is involved in this regulation. Incomplete reversal by either inhibitor implies that DLX6-AS1 regulates necroptosis via multiple signaling mechanisms. These findings clarify the pro-necroptotic mechanism of DLX6-AS1, underscore the complexity of its regulation, and necessitate further exploration of other involved pathways.

The PI3K/AKT/mTOR pathway is involved in cell oxidative stress, metabolism, proliferation, and apoptosis of cardiomyocytes, which affects myocardial ischemia‒reperfusion injury⁴³. While the PI3K/AKT pathway is well established as a cardioprotective cascade under physiological or transient stress⁴⁴, emerging evidence indicates that its sustained, maladaptive activation under pathological conditions may elicit detrimental outcomes. In myocardial ischemia/reperfusion (I/R) injury, mounting reports have revealed that hyperactivation of the PI3K/AKT/mTOR axis drives metabolic perturbation, inflammatory overactivation and regulated necrosis, rather than exerting a cytoprotective effect^45,46. In the current study, DLX6-AS1 overexpression triggered a persistent elevation in the phosphorylation levels of PI3K, AKT and mTOR in response to hypoxia/reoxygenation (H/R) stimulation. This molecular event was coupled with exacerbated cellular damage and enhanced necroptosis, as evidenced by upregulated MLKL phosphorylation. Notably, pharmacological blockade of PI3K signaling via LY294002 significantly alleviated DLX6-AS1-mediated necroptosis, thus verifying that DLX6-AS1 exacerbates myocardial injury by inducing maladaptive activation of the PI3K/AKT/mTOR pathway. Our findings are consistent with prior investigations, which have demonstrated that overactivation of the AKT/mTOR pathway during I/R impairs autophagic flux, aggravates mitochondrial dysfunction and accelerates necroptotic and inflammatory cell death. Collectively, these observations underscore the context- and stress intensity-dependent properties of AKT signaling, offering a mechanistic interpretation for the seemingly contradictory phenomenon of AKT activation coinciding with augmented cell death observed in this study.

Upregulation of DLX6-AS1 further attenuated cell viability and further increased LDH levels, indicating that cardiomyocyte injury was aggravated, the rate of apoptotic cells was elevated, and necroptosis-associated factors were elevated significantly. Importantly, phosphorylation of MLKL (p-MLKL), a key execution marker of necroptosis, was significantly increased following H/R injury and further enhanced by DLX6-AS1 overexpression, indicating activation of the necroptotic pathway. We proved that the above manifestations could function through the PI3K/AKT/mTOR pathway. After silencing DLX6-AS1, the above manifestations improved substantially, and the PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR levels decreased. In our in vitro assays, PI3K inhibitor administration partially mitigated DLX6-AS1 overexpression-induced necroptosis. This observation demonstrates that the PI3K/AKT/mTOR pathway is responsible for mediating a subset of DLX6-AS1’s pro-necroptotic actions, with other signaling cascades potentially contributing to this process. These findings underscore the intricate nature of DLX6-AS1-modulated necroptosis and imply the necessity of further studies to uncover additional implicated pathways. We demonstrated that DLX6-AS1 affects the necroptosis process of cardiomyocytes through the PI3K/AKT/mTOR pathway.

This study is subject to limitations. Direct transcriptomic data of DLX6-AS1 in human myocardial ischemia/reperfusion tissues are insufficient, and public human cardiac datasets do not contain lncRNA annotations for DLX6-AS1. This deficiency restricts the availability of direct human tissue evidence to support the clinical translational relevance of our conclusions. Subsequent research ought to fill these research gaps to strengthen the reliability and clinical applicability of our results.

Conclusion

We showed that DLX6-AS1 accelerated the progression of necroptosis and exacerbated MIRI, influenced by the PI3K/AKT/mTOR signaling pathway (Fig. 7). Evidence from our study suggests that targeting lncRNA DLX6-AS1 may offer a novel therapeutic approach for managing myocardial ischemia/reperfusion injury.

Abbreviations

DLX6-AS1

Distal-less homeobox 6 antisense 1

H/R

Hypoxia-reoxygenation

LAD

Left anterior descending coronary artery

MIRI

Myocardial ischemia/reperfusion injury

MLKL

Mixed lineage kinase domain-like protein

Nec-1

Necrostatin-1

RIP1/RIPK1

Receptor-interacting protein kinase 1

RIP3/RIPK3

Receptor-interacting protein kinase 3

Author contributions

Jun Zhang is responsible for sorting out the ideas of the project; Yanhong Su is responsible for animal experiments and paper writing; Liping Mu and Tian Li are responsible for cell experiments; Lei Wang is responsible for data collection; and Qianyu Zhang is responsible for statistical analysis.

Funding

Self-funded Project of Cangzhou Science and Technology Program (23244001006).

Data availability

All the data in the study are available upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Ethical approval was obtained from the Ethics Committee of Cangzhou Central Hospital (2024-029-01). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All efforts were made to minimize animal suffering and reduce the number of animals used.

Consent for publication

This manuscript has not been published elsewhere in part or in entirety and is not under consideration by another journal. All the authors have approved the manuscript and agree with submission to your esteemed journal.