Eight-week-old male C57BL/6 mice were provided by the Hangzhou Medical College. The mice were housed under standard conditions, with a temperature maintained at 22°C ± 1°C, humidity at 55% ± 5%, and a light-dark cycle of 12 h each. The mice were provided ad libitum access to food and water until sacrifice. All experimental procedures adhered to the guidelines approved by the Ethics Committees of Hangzhou Medical College (2018-045).

Sixty mice were deprived of food but provided with water normally for 4 h prior to streptozotocin (STZ, Sigma-Aldrich, United States, V900890) treatment, from 7 a.m. to 11 a.m. At 11 a.m., the mice received intraperitoneal injections of STZ at a dose of 50 mg/kg body weight per day, which dissolved in a 10 mmol/L citrate buffer, for five consecutive days to induce diabetes. The control (Ctl) mice were treated with an equivalent volume of citrate buffer only. One week later, the animals underwent a 4-hour fasting period, during which the treatment group’s fasting blood glucose concentrations were measured using a glucometer (LifeScan, United States). Animals with fasting blood glucose concentration higher than 16.7 mmol/L were considered as successful establishment of diabetes model. The mice were maintained for a duration of 12 weeks following STZ injection with recording the fasting blood glucose, body weight, and cardiac function.

The lncMEG3 short hairpin RNA (shRNA) was inserted into the heart-targeted adeno-associated virus 9 vector (cTNTp-EGFP-MIR155(MCS)-WPRE-SV40 PolyA, Genechem, China) (Chen et al., 2024). The AAV9 vector contained cTnT (cardiac-specific troponin T) promoter. And AAV9-cTnT-GFP was served as negative control. The virus was suspended in 0.9% NaCl solution with a viral titer of 1E+12 vg/mL, and subsequently, 200 μL of AAV9-shMEG3 were administered via tail vein injection to diabetic mice. Cardiac function and pathological changes were evaluated 12 weeks post-diabetes induction. The mouse lncMEG3 shRNA sequences are: 5′- ATT​CCA​GAT​GAT​GGC​TTT​GGC​GTT​TTG​GCC​ACT​GAC​TGA​CGC​CAA​AGC​TCA​TCT​GGA​AT-3′.

VINNO 6 Imaging System (VINNO, China) was employed for the assessment of cardiac function. The M-mode images of the left ventricle (LV) were recorded over three consecutive cardiac cycles. These images were used to calculate LV end-diastolic septal thickness (IVSd), LV end-systolic septal thickness (IVSs), LV end-diastole internal dimension (LVIDd), LV end-systolic internal dimension (LVIDs), LV end-diastole posterior wall thickness (LVPWd), LV end-systolic posterior wall thickness (LVPWs), ejection fraction (EF), and shortening fraction (FS).

The heart tissues were freshly collected, fixed in 4% paraformaldehyde (Biosharp, China) for 24 h at room temperature. After dehydration, the hearts were embedded with paraffin, and sectioned into 5 μm thin slices. Sections were imaged at ×400 magnification using light microscopy (CKX53, Olympus, Japan). Heart architectures were observed for myocardial hypertrophy in transverse heart sections. Utilizing ImageJ software (NIH, United States), quantification of 5 images per section was performed to measure the cardiomyocyte area (μm²) and determine myocardial hypertrophy.

The heart tissues were freshly collected, fixed in 4% paraformaldehyde (Biosharp, China) for 24 h at room temperature. After dehydration, the hearts were embedded with paraffin, and sectioned into 5 μm thin slices. The heart sections stained with a Masson’s trichrome were examined using a microscope (CKX53, Olympus, Japan) at ×200 magnification, and the collagen deposition of myocardial interstitium and perivascular was identified as blue staining, which is indicative of the presence of fibrotic tissue. The myocardial fibrotic area was analyzed using Image-Pro Plus 5.0 software.

The human cardiomyocyte line AC16 cardiomyocytes were purchased from Otwo biotechnology company Limited in Shenzhen, China. The AC16 cardiomyocytes were cultured in DMEM (HyClone, United States) medium supplemented with 5.5 mmol/L glucose and 10% bovine serum (BI, Israel), and maintained at a temperature of 37°C in a humidified atmosphere containing 5% CO₂. Following cell transfection, the AC16 cardiomyocytes were exposed to either a normal glucose medium (5.5 mmol/L) or a high glucose (HG) medium (35 mmol/L) for 24 h prior to subsequent experimental procedures.

LncMEG3 shRNA and miR-223 inhibitor (AMO-223) were designed and synthesized by GenePharma (GenePharma, China). The transfection of lncMEG3 shRNA and AMO-223 into AC16 cardiomyocytes was conducted using Lipofectamine 2000 transfection reagent (Invirogen, United States). The shRNA and AMO-223 were diluted in Opti-MEM medium and transfected into the cells. Following a 6-hour transfection period, the DMEM medium was replaced, and cell culture was continued for an additional 48 h.

The AC16 cardiomyocytes were seeded at a density of 1.0 × 10⁵ cells per well in a 12-well plate to ensure even distribution, followed by cell transfection, exposure to high glucose, and other treatments according to the experimental groups. Subsequently, the cells were fixed using 4% paraformaldehyde (Beyotime, China) for 15 min at room temperature. After that, 1.5 mL of 0.5% Triton X-100 (Beyotime, China) was added to each well for permeabilization and incubated for 15 min. Subsequently, the fixed cells were blocked with 5% bovine serum albumin (BSA, Beyotime, China) at room temperature for a duration of 1 h, followed by overnight incubation on a shaker at 4°C with diluted α-actin antibody (Abcam, ab137346, United Kingdom). The next day, incubate the Alexa Fluor^® 594 fluorescent secondary antibody (abcam, ab150080, United Kingdom) at room temperature for 2 h and then add Hoechst 33258 (Beyotime, China) diluted with PBS to a final concentration of 5 μg/mL for 5 min. The entire process was shielded from light, and the cellular morphology was observed and captured using a confocal microscope (CytationM, BioTek, United States).

Total RNA was isolated utilizing TRNzol reagent (Tiangen, China), followed by quantification of concentration and assessment of purity using a Nanodrop nucleic acid and protein analyzer (Thermo Fisher Scientific, United States). After quantifying the RNA concentration, 1,000 ng of the RNA was employed for reverse transcription utilizing the PrimeScript RT Reagent Kit (Takara, China). The RNA expression level was detected using the QuantStudio 3 real-time PCR system (Applied Biosystems, United States) with SYBR Green-based real-time PCR analysis. GAPDH was used as the internal control in this study, and all gene expression levels were calculated using 2^−△△CT method. The real-time PCR was conducted according to the following temperature protocol: 95°C for 30 s and then 40 cycles of 95°C for 10 s, 60°C for 20 s, 72°C for 20 s. The RNA primers were procured from Thermo Fisher Scientific (Thermo, China) and the sequences are provided in Supplementary Table S1 in the Supplementary Material.

AC16 cardiomyocytes were lysed with 200 μL of RIPA protein lysis buffer per group (Beyotime, China) to extract total protein. Mice myocardial tissue was lysed with 300–400 μL of RIPA buffer to extract total protein. The protein lysates, containing 60 μg of protein, were subjected to SDS-PAGE for separation. Subsequently, they were transferred onto nitrocellulose membranes (Millipore, United States) and subsequently blocked using a solution consisting of 2.5% BSA (Beyotime, China) and 2.5% non-fat milk (BD, United States). The membranes were incubated with the primary antibody overnight at 4°C, followed by a subsequent incubation with the secondary antibody for 1 hour at room temperature on the following day. The immunoblot was tested and quantified by the Image Studio (LI-COR Biosciences, United States). The primary antibodies were used as follows: Atrial natriuretic peptide (ANP, Abcam, ab262703, United Kingdom), Brain natriuretic peptide (BNP, Abcam, ab236101, United Kingdom), NLRP3 (HuaAn Biotechnology, ET1610-93, China), Apoptosis-associated speck-like protein containing a CARD (ASC, Cell Signaling Technology, 67824 and 13833, United States), Caspase-1 (Cell Signaling Technology, 8338, United States), Cas-1 cut (Cell Signaling Technology, 4199, United States), IL-1β (Cell Signaling Technology, 12242, United States), IL-18 (Cell Signaling Technology, 57058 and 54943, United States), and GAPDH (Bioker Biotechnology, BK7021, China).

The statistical analyses were conducted using SPSS 22.0, and the results were presented as mean ± SD. Statistical analysis of all data was performed using One-Way ANOVA followed by the Tukey test, with statistical significance defined as P < 0.05. The gray value of Western blotting immunobands was analyzed using Odyssey 4.0 software.

To investigate the involvement of lncMEG3 in DCM, we initially assessed the expression levels of lncMEG3 in DCM mouse hearts and HG-treated AC16 cardiomyocytes. The expression of lncMEG3 was significantly upregulated in diabetic mice heart and AC16 cardiomyocytes treated with HG (Figures 1A, B). To further validate the roles of lncMEG3 in DCM, the interfering RNA molecules targeting lncMEG3 were synthesized and added into diabetic mice and AC16 cardiomyocytes to assess their inhibitory potential on lncMEG3 expression. The results demonstrated that shMEG3 exhibited a significant effect (Figures 1C, D).

To elucidate the functional role of lncMEG3 in DCM, a heart-targeted adeno-associated virus carrying lncMEG3 interfering RNA (AAV9-shMEG3) was administered via tail-vein injection into diabetic mice. After STZ injection, the mice exhibited an elevated level of fasting blood glucose (FBG) and a reduction in body weight at various time points compared to control mice. The silencing of lncMEG3 resulted in a significant reduction in fasting blood glucose (FBG) levels at the 12-week time point (Figure 1E). No significant differences were observed in FBG levels at other time points between the diabetic mice and lncMEG3 knockdown diabetic mice. The knockdown of lncMEG3 did not result in any statistically significant differences in the body weight of diabetic mice (Figure 1F). No significant differences were observed in heart weight, liver weight, and kidney weight among the mice within each group (Figure 1G). However, compared to the control group, diabetic mice exhibited a significantly increased heart weight/body weight ratio, liver weight/body weight ratio, and kidney weight/body weight ratio. Notably, silencing lncMEG3 resulted in a significant reduction in the heart weight/body weight of diabetic mice (Figure 1H).

Echocardiography was conducted on mice to investigate the impact of lncMEG3 silencing on cardiac function in diabetic murine models. The results showed that ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic septal thickness (IVSd), left ventricular end-systolic septal thickness (IVSs), left ventricular end-diastole internal dimension (LVIDd), left ventricular end-systolic internal dimension (LVIDs), left ventricular end-diastole posterior wall thickness (LVPWd), and left ventricular end-systolic posterior wall thickness (LVPWs) were significantly deteriorated in diabetic mice compared with the control group (Figures 2A–E). However, silencing of lncMEG3 mitigated the decline in EF, FS, and IVSs, while preventing the increase in LVIDs and LVIDd in diabetic mice (Figures 2A–D). To investigate the impact of lncMEG3 silencing on cardiomyocyte hypertrophy and cardiac fibrosis in diabetic mice, we employed H&E staining and Masson staining to examine pathological alterations in murine hearts. The diabetic group exhibited a significant increase in the area of cardiomyocytes, myocardial interstitial fibrosis, and perivascular fibrosis, as depicted in Figures 2F,G. Conversely, silencing lncMEG3 expression in cardiomyocytes of mice remarkably reduced the aforementioned areas in diabetic mice. Notably, the negative control group did not exert a significant effect on cardiac remodeling.

To investigate the effect of lncMEG3 on DCM, we assessed the level of myocardial hypertrophy-associated proteins in DCM mouse heart. ANP, BNP and β-MHC were utilized as markers of cardiac hypertrophy to evaluate the occurrence of myocardial hypertrophy in DCM mice. Our findings revealed a significant upregulation of ANP, BNP, and β-MHC expression in DCM mice. Moreover, lncMEG3 knockdown reduced the levels of myocardial hypertrophy-related genes (Figures 3A–C). Then, we explored the regulation of lncMEG3 on cardiac hypertrophy induced by HG in vitro. The results demonstrated that myocardial cell area was increased in HG-treated AC16 cardiomyocytes, with elevated protein levels of ANP and BNP. However, knockdown of lncMEG3 ameliorated these phenomena (Figures 3D–F), indicating that lncMEG3 deficiency significantly attenuated the deterioration of myocardial hypertrophy in DCM.

The impact of lcnMEG3 deficiency on myocardial pyroptosis was investigated by assessing the expression levels of key factors associated with the NLRP3/Caspase-1/IL-1β classical pyroptosis signaling pathway in murine cardiac tissue. As shown in Figures 4A–F, the protein expressions of NLRP3, ASC, Caspase-1 cut, Caspase-1, IL-1β, and IL-18 were found to be elevated in the hearts of diabetic mice compared to the control group. While, the silencing lncMEG3 resulted in a significant reduction in expression levels of pyroptosis signaling factors. The mRNA levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the heart tissues of different mouse groups were quantified through real-time PCR. It was found that compared with the DCM group, silencing lncMEG3 significantly attenuated the mRNA levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the hearts of diabetic mice (Figures 4G–K). These findings suggest that silencing lncMEG3 can effectively inhibit the pyroptosis signaling pathway in diabetic mouse hearts.

We subsequently investigated the effect of lncMEG3 on pyroptosis signaling pathway treated by HG (35 mmol/L) in vitro. The exposure to HG significantly facilitated activation of pyroptosis signaling pathway. The level of lncMEG3 was reduced in cultured AC16 cardiomyocytes through transfection with lncMEG3 shRNA. LncMEG3 knockdown resulted in a decrease in the mRNA and protein levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 (Figure 5), indicating that silencing lncMEG3 effectively inhibit the activation of pyroptosis signaling pathway in AC16 cardiomyocytes.

We next continued to explore how lncMEG3 regulate pyroptosis signaling pathway in HG-treated AC16 cardiomyocytes. Through RNAhybrid web analysis, we predicted potential microRNA targets of lncMEG3 and identified a binding site for miR-223 within lncMEG3 (Figure 6A). We quantified the expression levels of miR-223 through real-time PCR and observed a significant decrease in miR-223 RNA levels in DCM mouse and AC16 cardiomyocytes treated with HG (Figures 6B, C). LncMEG3 shRNA dramatically increased the expression of miR-223 (Figures 6D, E). Several studies have reported that NLRP3 expression is regulated by miR-223 (Zhang et al., 2018).

LncMEG3 shRNA and AMO-223 (miR-223 inhibitor) were co-transfected into AC16 cardiomyocytes to further elucidate the role of lncMEG3 in promoting cardiomyocyte hypertrophy by regulating miR-223. Therefore, α-acting staining and assessment of hypertrothy-related gene expression were used to evaluate myocardial hypertrophy. As shown in Figures 6F–H, the area of cardiomyocytes and the expressions of ANP and BNP were significantly upregulated by AMO-223. These findings suggest that lncMEG3 may promote myocardial hypertrophy by inhibiting the level of miR-223.

To elucidate the involvement of lncMEG3/miR-223 in the regulation of the NLRP3 inflammasome-mediated pyroptosis signaling pathway, we employed a co-transfection approach to inhibit the lncMEG3/miR-223 axis by transfecting lncMEG3 shRNA and AMO-223 into AC16 cardiomyocytes under high glucose conditions. The real-time PCR analysis revealed that the expression of miR-223 in AC16 cardiomyocytes stimulated by HG was significantly downregulated compared to the negative control group. However, knockdown of lncMEG3 in HG-treated AC16 cardiomyocytes resulted in a significant upregulation of miR-223 expression. Conversely, co-transfection of lncMEG3 shRNA and AMO-223 led to a reduction in miR-223 expression. The protein expression levels of factors associated with the NLRP3 inflammasome-mediated pyroptosis signaling pathway in AC16 cells were assessed by Western blotting. As depicted in Figure 7, compared to the HG group, knockdown of lncMEG3 shRNA and stimulation with HG significantly decreased the expressions of pyroptosis-related proteins including NLRP3, ASC, Caspase-1, Caspase-1 cut, IL-1β, and IL-18. Conversely, co-transfection of lncMEG3 shRNA and AMO-223 upregulated the expression of these pyroptosis-related proteins. These findings suggest that silencing lncMEG3 inhibits the cell pyroptosis pathway through miR-223 upregulation, thereby ameliorating diabetic cardiomyopathy.