Twenty female and ten male Sprague-Dawley rats (250–300 g) were raised at the Animal Center of the Qingdao University Medical College. The rats received regular feeding and were exposed to natural light and a room temperature of 19–24°C. After acclimation for a few days, the rats were caged according to a 2:1 ratio of males to females. Vaginal monitoring was performed the next morning; vaginal plug formation was considered day 1 of pregnancy. Pregnant rats were randomly divided into the following groups (n = 5): low FA exposure (0.2 mg/kg), medium FA exposure (2 mg/kg), high FA exposure (20 mg/kg) (29), and control group (normal saline). On the 7th day of pregnancy, the appropriate concentration of FA (or saline) was injected into the abdominal cavity once daily for 12 days. The pregnant rats were closely monitored and anesthetized via intraperitoneal injection of chloral hydrate (0.3 mL/100 g) on the 12th day of treatment exposure.

The pregnant rats were randomly divided into three groups (n = 5, in vivo recovery experiment): control, medium FA concentration, and medium FA concentration together with miR-153-3p antagomir. Animal breeding conditions are as described above. From the first day to the fourth day of FA treatment, miR-153-3p antagomir (2 mg/kg) was injected into the rat tail vein every day. The pregnant rats were closely monitored and anesthetized via intraperitoneal injection of chloral hydrate (0.3 mL/100 g) on the 12th day of FA exposure. Fetal rat hearts were then collected, and tissue samples were either paraffin-sectioned and stained or homogenized for total RNA and total protein extraction. The Research Ethics Committees of the Affiliated Hospital of Qingdao University approved this study, and all experiments were conducted following the principles of the Declaration of Helsinki.

Rat embryonic cardiomyocyte cell line H9C2 (American Type Culture Collection, Manassas, VA, USA) was routinely resuscitated using DMEM complete medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (ExCell Bio, Shanghai, China). Cells were cultured in a constant temperature incubator at 37°C under 5% CO₂ until reaching 95% confluency. Cells were transfected with Lipofectamine 3,000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. miR-153-3p mimic (GenePharma, Shanghai, China): 5′-UUGCAUAGUCACAAAAGUGAUC−3′, 5′-UCACUUUUGUGACUAUGCAAUU-3′; mimic NC (GenePharma): 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′. miR-153-3p inhibitor (GenePharma): 5′-GAUCACUUUUGUGACUAUGCAA−3′; inhibitor NC (GenePharma): 5′-CAGUACUUUUGUGUAGUACAA−3′. Si-βII spectrin: 5′-GGACAUGUCUUAUGAUGAATT-3′, 5′-UUCAUCAUAAGACAUGUCCTT-3′; siRNA control: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCG GAGAATT-3′ (GenePharma). For the FA induction experiment, H9C2 cells were treated with 0, 50, 100, and 150 μmol/L FA (Sigma-Aldrich, St. Louis, MO, USA) for different times (0, 1, 3, 6, 12, and 24 h).

TRIzol reagent (Invitrogen) was used to extract RNA from H9C2 cells and fetal heart tissues. Additionally, tissue samples were cut, homogenized, and mixed with chloroform prior to RNA isolation. The precipitate was thoroughly washed with 75% ethanol prepared with DEPC-treated water and finally dried to obtain isolated RNA. After measuring the concentration, reverse transcription of RNA into cDNA is done by PrimeScript Reverse Transcription (RT) reagent kit (Takara Bio, Kyoto, Japan). Hieff UNICON Power qPCR SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China) was used for RT-qPCR (Bio-Rad, American), with GAPDH used as the internal reference. All experimental steps were performed in accordance with the manufacturer's instructions. Amplification conditions: denaturation: 95°C, 5 min; annealing: 60°C, 30 s; extension: 95°C, 5 s; 40 cycles. The PCR primers are shown in Table 1.

Cells were lysed with an appropriate amount of RIPA lysis buffer (Solarbio, Beijing, China) for 10 min on ice. The mixture was then centrifuged, and bicinchoninic acid (BCA; Solarbio) method determines protein concentration, employing a microplate reader to detect the absorbance of the protein sample at 562 nm. Proteins were separated by 7–12.5% SDS-PAGE (Solarbio), and transferred to polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk. Then, it incubated with primary antibody (βII spectrin, Abcam, ab72239; Cleaved caspase 7, Cell Signaling Technology, #8438S; Caspase 7, Abclonal, A1524; Bax, Abclonal, A19684; Bcl 2, Zenbio, 250414), followed by secondary antibody (Abcam, Cambridge, UK). Proteins were detected using the Fusion Solo S system (Vilber, Paris, France).

Fetal heart tissue was fixed in 4% FA solution and paraffin-sectioned. Sections were then deparaffinized in xylene, rinsed with PBS, and incubated in 3% H₂O₂ in 50% methanol at 37°C for 30 min. After eliminating endogenous peroxidase activity, and incubated in a protein blocking solution (Bio-Genex, San Ramon, CA, USA) for 30 min. Sections were incubated with βII spectrin antibody (1:100; ab72239, Abcam) overnight at 4°C to block non-specific binding. Then, goat anti-rabbit secondary antibody for 30 min. The peroxidase substrate diaminobenzidine was used for the reaction. Hematoxylin counterstain, observed under a microscope (Nikon, Tokyo, Japan).

Fetal heart tissue was embedded, frozen, sectioned, and FISH was performed to detect the expression of miR-153-3p, as previously described (30). The sequence of the miR-153-3p probe (GenePharma) used for FISH was 5′-CY5-GATCACTTTTGTGACTATGCAA-3′; NC probe: 5'-CY5-TTAGAGGCATCTCGTA ATCTAT-3′. Nuclei were stained using DAPI and slides were mounted using Vectashield mounting agent (Vector Labs, Burlingame, CA, USA).

H9C2 cells were seeded into a 10 cm dish. The cells were subsequently scraped off and a glass grinder was used to fully lyse the cells on ice. Centrifuge at low temperature and collect the supernatant for further use. The miR-153-3p probe (GenePharma) sequences were: miR-153-3p WT 5′-bio- UUGCAUAGUCACA AAAGUGAUC-3′, miR-153-3p Mut 5′-bio- AACGUAUGUCACAAAAGUGAUC-3′. The scrambled control probe was: 5′-bio- AUAAGUACUGUAGUAGAACUCC-3′. The probe was dissolved according to the manufacturer's protocol, and 10 μL of the dissolved probe solution was mixed with 30 μL Pierce Streptavidin Agarose (Thermo-Fisher Scientific, Paisley, UK) and treated with buffer (31). Finally, the lysed sample was mixed with the Pierce Streptavidin Agarose-probe solution, and incubated overnight at 4°C. TRIzol reagent was added to extract RNA, which was then used for βII spectrin detection by RT-qPCR.

The reconstructed pmirGLO luciferase vector (GenScript Biotech, Piscataway, NJ, USA) contained the 3′UTR fragment of wild-type βII spectrin (βII spectrin-Wt) with a miR-153-3p binding site or that of mutant βII spectrin without a miR-153-3p binding site (βII spectrin-Mut). To measure luciferase activity, 293T cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were seeded into a 24-well plate. Use Lipofectamine 3,000 reagent to co-transfect with reconstituted luciferase vector and miR-153-3p mimic or NC mimic. After 48 h of transfection, use Dual-Luciferase Reporter Gene Assay kit (Meilunbio, Dalian, China) to detect luciferase activity according to the manufacturer's instructions.

H9C2 cells were seeded into a 24-well plate and cardiomyocyte apoptosis was evaluated under different conditions using TUNEL assay, performed using the TUNEL Apoptosis Detection kit (Yeasen Biotechnology Co., Ltd.) according to the manufacturer's instructions. Cells in each well were incubated with 50 μL TUNEL mixture at 37°C for 1 h. Stain cell nuclei using DAPI. The apoptotic rate was determined as the number of apoptotic cells (red)/total number of cells (blue) × 100%. Cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan). The optical density was measured using ImageJ v1.5.1 software.

Cell viability was measured using Cell Counting Kit-8 (Yeasen Biotechnology Co., Ltd.). A total of 4 × 10³ H9C2 cells per well were seeded into a 96-well plate. According to the manufacturer's instructions, 10 μL CCK-8 solution was added to 100 μL medium. Reaction conditions: 37 °C, 2 h. The absorbance (Bio-tek, American) was measured at 450 nm.

Apoptosis was evaluated using the Annexin V-FITC Cell Apoptosis Detection kit (Meilunbio). H9C2 cells were subsequently collected, treated with trypsin without EDTA, washed with PBS, and finally centrifuged. According to the manufacturer's instructions, the cell pellet was resuspended in binding buffer and incubated with annexin V-FITC reagent and propidium iodide in the dark. Finally, the cells were analyzed using a Beckman FC 400 MPL flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

Fetal heart tissue was fixed with 10% FA at room temperature for 24 h, then decalcified, dehydrated, permeated with xylene, embedded in wax. Wiegert's iron hematoxylin solution (Sigma-Aldrich) was used to stain the nuclei for 5 min. First stain with 0.7% Masson's Trichrome Stain solution (Sigma-Aldrich) for 10 min after rinsing with distilled water. Rinsing: 2% glacial acetic acid; differentiation: in phosphomolybdic acid, 4 min. Continue dyeing with 2% aniline blue dye solution (Sigma-Aldrich). After dehydration, dewaxing, and fixation with neutral resin, the image was taken with an optical microscope (Nikon).

Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA) and IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA) software. The results are expressed as mean ± standard deviation values. Statistical differences were determined by unpaired Student's t-test, one-way analysis of variance (ANOVA), and Tukey's post- hoc test or Mann-Whitney test, depending on the distribution of variables. A p-value < 0.05 was considered statistically significant. Each experiment was repeated at least three times.

The role of βII spectrin in embryogenesis, especially in heart development, has been partially determined in previous studies. Specifically, βII spectrin is essential for the survival and differentiation of cardiomyocytes, and its loss can lead to defects in cardiac development and the inability to thicken the ventricular wall (32). In addition, FA is closely associated with apoptosis (33, 34). At present, whether βII spectrin is involved in the molecular mechanism underlying FA-induced fetal heart development defects remains unclear. Immunohistochemical analysis revealed that βII spectrin expression was reduced in fetal rat hearts after intraperitoneal injection of FA (Figure 1A). According to previous studies (35, 36), FA can induce apoptosis in lung cells and mouse bone marrow cells. In our study, with the extension of the treatment time, the concentration of 150 μmol/L FA also significantly reduced the number of H9C2 cells (Figure 1B). RT-qPCR analysis revealed that FA did not significantly affect the expression of proliferation-related protein PCNA (proliferating cell nuclear antigen) (Figure 1C). Furthermore, we found that at a concentration of 150 μmol/L FA, the expression of apoptosis-related proteins Bax and caspase7 increased in H9C2 cells, while that of apoptosis-related Bcl-2 decreased (Figure 1C). Western blotting revealed that the expression of cleaved caspase7 increased significantly when cells were treated with 150 μmol/L FA, whereas the expression of caspase7 decreased significantly (Figure 1D). Immunohistochemical analysis revealed that caspase7 expression was reduced in fetal rat hearts after intraperitoneal injection of FA (Figure 1E). Further RT-qPCR analysis indicated that βII spectrin expression gradually decreased with time when cells were treated with 150 μmol/L FA, which was confirmed by western blotting (Figures 1F,G). Based on these results, we speculate that certain FA concentrations can lead to increased cardiomyocyte apoptosis, which might lead to the onset and progression of CHD.

We employed bioinformatics analysis to predict the upstream miRNA of βII spectrin in rats using the TargetScan and miRWalk databases to identify 103 miRNAs, shown in the Venn diagram. We screened conserved miRNAs reportedly related to cardiovascular disease and apoptosis in the literature, and those displaying significant changes in expression when cells were treated with 150 μmol/L FA (Figure 2A). Among them, the expression of miR-153-3p increased most significantly (Figure 2B). Notably, miR-153-3p is highly conserved in humans, rats, and mice (Figure 2C). In order to further verify whether the in vitro miR-153-3p results were consistent in the animal model, FISH was used to detect the expression of miR-153-3p in heart tissues of fetal rats treated with different FA concentrations (0.2 mg/kg and 2 mg/kg). Consistently, miR-153-3p expression was increased in the FA-treated groups, with the greatest increase observed in the 2 mg/kg treatment group (Figure 2D). We speculate that miR-153-3p is essential in the regulatory pathway responsible for FA-induced reduction of βII spectrin expression.

To further verify the interaction between miR-153-3p and βII spectrin in vitro, we transfected H9C2 cells with the miR-153-3p mimic or inhibitor with high transfection efficiency (Figure 2E). RT-qPCR was employed to detect changes in βII spectrin expression in H9C2 cells after transfection, indicating that βII spectrin expression in the miR-153-3p mimic-transfected group was lower than that in the control group. However, βII spectrin expression in the miR-153-3p inhibitor-transfected group was increased (Figure 2F). We also verified these results at the protein level by WB (Figure 2G). Moreover, the interaction between miR-153-3p and βII spectrin was further verified under pathological stimulus conditions, revealing that transfection with the miR-153-3p inhibitor significantly restored the FA-induced decrease in βII spectrin expression (Figure 2H). Consistently, βII spectrin expression was reduced in cells treated with the miR-153-3p mimic and FA (Figure 2I). We speculated that miR-153-3p directly interacts with βII spectrin. Therefore, the RNA pull-down assay was performed using the miR-153-3p biotin probe, indicating that βII spectrin significantly bound to miR-153-3p at the mRNA level, compared with the control group (Figure 2J). In order to further verify the possibility of direct binding between βII spectrin and miR-153-3p, we co-transfected 293T cells with the reconstructed luciferase vectors (with βII spectrin-Wt and βII spectrin-Mut 3'UTR) and the miR-153-3p mimic (Figure 2K). The luciferase activity of 293T cells co-transfected with βII spectrin-Wt 3'UTR and the miR-153-3p mimic was significantly lower than that of cells co-transfected with βII spectrin-Mut 3'UTR and the miR-153-3p mimic (Figure 2L).

We explored the effect of miR-153-3p overexpression on cardiomyocyte function by transfecting H9C2 cells with the miR-153-3p mimic and examining cell behavior at 0, 12, 24, and 36 h. The CCK-8 assay revealed that cell proliferation was not significantly affected by transfection with the miR-153-3p mimic (Figure 3A). Previous research has shown that α-SMA, dystrophin, and NKX2.5 are cardiomyocyte differentiation markers, and PCNA is associated with proliferation. Therefore, we transfected H9C2 cells with the miR-153-3p mimic for 24 h and assessed the expression of the above-mentioned markers. RT-qPCR analysis indicated that expression changes in the differentiation and proliferation markers were not significant (Figure 3B). Bax, Bcl-2, and Caspase7 have been previously associated with apoptosis in liver cancer, and βII spectrin expression is closely related to that of caspase7. Therefore, we measured the expression of Bax, Bcl-2, and caspase7 after transfecting H9C2 cells with the miR-153-3p mimic for 24 h. The results demonstrated that Bax and caspase7 expression increased significantly, while that of Bcl-2 decreased significantly (Figure 3C). The TUNEL assay results confirmed that transfection with the miR-153-3p mimic for 24 h significantly increased the apoptosis of H9C2 cells compared with the control group (Figure 3D). We then stimulated the transfected cells with 150 μmol/L FA for 24 h and performed cell flow cytometry experiments. Consistently, apoptosis rate was strongly activated after transfection of miR-153-3p mimics (11.62%) than NC group (4.67%) as well as FA induced condition (Figure 3E). The TUNEL assay results confirmed these findings (Figure 3F). Taken together, the results demonstrate that overexpression of miR-153-3p promoted the apoptosis of H9C2 cells.

We further explored the effect of miR-153-3p knockdown on cardiomyocyte function. Assessing cell proliferation at 0, 12, 24, and 36 h after transfecting H9C2 cells with the miR-153-3p inhibitor, the CCK-8 assay results indicated that cell proliferation was not significantly affected by transfection with the miR-153-3p inhibitor (Figure 4A). Consistently, transfecting H9C2 cells with the miR-153-3p inhibitor for 24 h did not significantly change the expression of the differentiation and proliferation markers, dystrophin, NKX2.5, and PCNA (Figure 4B). However, the expression of Bax and Caspase7 decreased, while that of Bcl-2 increased (Figure 4C). To further verify whether the miR-153-3p inhibitor inhibited apoptosis, TUNEL staining was employed to evaluate H9C2 cells transfected with the miR-153-3p inhibitor for 24 h. Compared with the control group, the miR-153-3p inhibitor-transfected group displayed fewer apoptotic cells (Figure 4D). Flow cytometry analysis of cells transfected with the miR-153-3p inhibitor for 24 h, followed by stimulation with 150 μmol/L FA for 24 h, revealed that transfection with the miR-153-3p inhibitor reversed FA-induced apoptosis (Figure 4E). These results were further confirmed by TUNEL staining (Figure 4F). Taken together, the results demonstrate that miR-153-3p knockdown inhibited the apoptosis of H9C2 cells.

We further verified βII spectrin targeting of miR-153-3p under pathological stimuli. Cells were co-transfected with the βII spectrin inhibitor and the miR-153-3p mimic or inhibitor for 24 h and then were stimulated with FA for 24 h. We co-transfected H9C2 cells with the βII spectrin inhibitor designed by GenePharma and the miR-153-3p mimic or inhibitor, employing RT-qPCR and western blotting to detect changes in the expression of βII spectrin. βII spectrin expression was significantly reduced when cells were co-transfected with the miR-153-3p mimic and the βII spectrin inhibitor, and treated by FA (Figures 5A,B). WB showed that cleaved caspase7 expression was significantly increased in the group treated with βII spectrin inhibitor, miR-153-3p mimic, and FA, and transfection with miR-153-3p inhibitor reversed this phenomenon (Figure 5C). TUNEL staining indicated that the highest level of apoptosis was observed in the group treated with the βII spectrin inhibitor, miR-153-3p mimic, and FA, while transfection with the miR-153-3p inhibitor reversed the apoptosis (Figure 5D). Cell flow cytometry experiments further verified the above results (Figure 5E). The above confirmed that miR-153-3p can target βII spectrin to regulate cardiomyocyte apoptosis.

The in vitro experiments investigated the interaction between miR-153-3p and βII spectrin when H9C2 cells were stimulated with FA. To further confirm these results in the animal model, we chose three FA concentrations for intraperitoneal injection based on previous research (29). During the experiment, the high FA concentration (20 mg/kg) resulted in a high fatality rate for pregnant rats and a low pregnancy rate; therefore, the low (0.2 mg/kg) and medium (2 mg/kg) FA concentration groups were selected for further analysis. Masson's trichrome staining revealed increased myocardial fibrosis in the FA treatment groups compared with the control group (Figure 6A). The RT-qPCR results indicated that βII spectrin expression decreased, while that of miR-153-3p increased in the FA treatment groups (Figures 6B,C). Western blotting revealed that βII spectrin expression decreased in the FA treatment groups (6D). In addition, Bax and caspase7 expression in the FA treatment group increased at the mRNA level, while that of Bcl-2 decreased, with significant changes observed in the group treated with 2 mg/kg FA (Figures 6E–G). At the protein level, the expression of Bcl-2 and caspase7 in the FA treatment group decreased, while that of cleaved caspase7 and Bax increased (Figure 6H). The in vivo results were consistent with those of the in vitro experiments, confirming that a certain concentration of FA promoted the occurrence of cardiomyocyte apoptosis.

To further verify the above results, we conducted an in vivo recovery experiment. The pregnant rats were divided into three treatment groups: control, medium FA concentration, and medium FA concentration plus miR-153-3p antagomir. As before, we used masson's trichrome staining revealed that compared with the FA treatment group, FA treatment and miR-153-3p antagomir group reversed myocardial fibrosis (Figure 6I). we used RT-qPCR to measure mRNA levels of βII spectrin and miR-153-3p expression in fetal heart tissues (Figures 6J,K). Western blotting indicated that βII spectrin expression in the medium FA concentration plus miR-153-3p antagomir group was recovered compared with that in the medium FA concentration group (Figure 6L). Immunohistochemical experiments also revealed that βII spectrin expression was restored (Figure 6M). Moreover, western blotting revealed that the expression of Bcl-2 and caspase7 in the medium FA concentration plus miR-153-3p antagomir group was recovered, while that of cleaved caspase7 and Bax decreased (Figure 6N). Immunohistochemical experiments also revealed that caspase7 expression was restored (Figure 6O). Altogether, the results indicated that miR-153-3p can directly target βII spectrin to regulate FA-induced cardiomyocyte apoptosis.