EA.hy926 cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Biological Industries; Sartorius AG) and 1% streptomycin/penicillin (Beyotime Institute of Biotechnology) at 37°C in a saturated humidity incubator containing 5% CO₂. When cell confluence reached 70–80%, the cells were starved in serum-free medium for 12 h to synchronize the cell cycle and were then incubated with rapamycin (cat. no. B20714; Shanghai Yuanye Biotechnology Co., Ltd.) at a concentration of 1,000 nM in a 5% CO₂ cell incubator at 37°C for 6 h to establish the autophagy model. The cells in the control group did not receive any treatment.

LC3I/LC3II, p62, Beclin1 and FOXO3 expression levels were determined using western blotting. Total protein was extracted from EA.hy926 cells using radioimmunoprecipitation assay buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and were quantified using a BCA kit (cat. no. P0009; Beyotime Institute of Biotechnology). A Bio-Rad iMark microplate reader (Bio-Rad Laboratories, Inc.) was used to measure the absorbance value of each sample at a wavelength of 540 nm. The protein concentration was calculated according to the standard curve, after which, equal samples of protein (20 µg) were separated by SDS-PAGE on 10 or 20% gels, and were transferred to a polyvinylidene fluoride membrane. After being blocked with 5% skim milk for 1 h at room temperature, the membrane was incubated with rabbit primary antibodies against LC3I/LC3II (cat. no. WL01506; 16/14 kDa), p62 (cat. no. WL02385; 55 kDa), Beclin1 (cat. no. WL02508; 60 kDa) and FOXO3 (cat. no. WL02891; 70 kDa) (all from Shenyang Wanlei Biotechnology Co., Ltd. and diluted to 1:1,000) at 4°C overnight. Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. WLA023; 1:5,000, Shenyang Wanlei Biotechnology Co., Ltd.) at 37°C for 1 h. Target bands were developed using a chemiluminescence substrate kit (cat. no. P0018S; Beyotime Institute of Biotechnology) and were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.). β-actin (cat. no. WL0002d; 43 kDa; Shenyang Wanlei Biotechnology Co., Ltd.; diluted to 1:1,000) was used as an internal reference.

Immunofluorescence analysis was performed to assess the expression of LC3B. The cells were fixed with 4% paraformaldehyde (cat. no. P0099; Beyotime Institute of Biotechnology) for 15 min at room temperature and permeabilized with 0.1% Triton X-100 (cat. no. P0096; Beyotime Institute of Biotechnology) for 15 min at room temperature. Goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) was used to block the sections for 30 min at room temperature. Subsequently, the cells were incubated with an anti-LC3B rabbit antibody (cat. no. WL01506; 1:200; Shenyang Wanlei Biotechnology Co., Ltd.) at 4°C overnight. The cells were then incubated with an AlexaFluor^® 594-conjugated goat anti-rabbit IgG secondary antibody (cat. no. RS3611; 1:1,000; ImmunoWay Biotechnology Company) at room temperature for 1 h. The nuclei were stained at room temperature with DAPI (1 µg/ml; cat. no. C1006; Beyotime Institute of Biotechnology) for 10 min. A Leica TCS SP laser scanning confocal microscope (Leica Microsystem GmbH) was used to obtain micrographs. The immunofluorescence intensity was measured using Image-ProPlus 6.0 software (Media Cybernetics, Inc.).

The treated cells were moved to a 1.5-ml centrifuge tube using a cell scraper and then centrifuged at 1,006.2 × g for 5 min at room temperature. Subsequently, the supernatant was discarded, and 1 ml 2.5% glutaraldehyde (Beyotime Institute of Biotechnology) was added to the sediment for fixation at 4°C for 4 h. The fixed samples were then washed with phosphate-buffered saline, dehydrated at 4°C with successively increasing percentages of acetone (50, 70, 90 and 100%; 15 min each) and soaked overnight at room temperature with Spurr's resin. Subsequently, the samples were incubated overnight in a 37°C oven and baked for 48 h in a 60°C oven. The samples were then cut into ultrathin slices (60–80 nm) using an ultrathin microtome (Leica Microsystems GmbH), stained with uranyl acetate for 15 min at room temperature, rinsed with distilled water, stained with lead citrate for 15 min at room temperature, rinsed again with distilled water and observed by TEM (Hitachi, Ltd.) after air-drying overnight.

The expression levels of miR-92a and FOXO3 were measured using RT-qPCR. Briefly, total RNA was isolated from EA.hy926 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RNA was then reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Kit (cat. no. AG11705; Accurate Biotechnology Co., Ltd.) or the miRNA 1st strand cDNA synthesis kit (cat. no. AG11717; Accurate Biotechnology Co., Ltd.), according to the manufacturer's protocols, and qPCR reaction was carried out according to the following amplification conditions: Pre-denaturation at 94°C for 2 min for one cycle; followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min; and a final extension step at 72°C for 6 min. The relative changes in mRNA expression were calculated using the 2^−ΔΔCq formula (29). Among them, the reverse miR-92a primer was obtained from its RT reagent kit (cat. no. AG11717; Accurate Biotechnology Co., Ltd.). GAPDH and U6 were used as internal controls for mRNA and miRNA, respectively, and the primers for qPCR were synthesized by General Biology (Anhui) Co., Ltd. with the following sequences: FOXO3, forward 5′-CTCTCTCGCCCATGCTCTAC-3′, reverse 5′-CCGAGCCCTTGGTGGTATA-3′; miR-92a, forward 5′-TATTGCACTTGTCCCGGCCTG-3′; GAPDH, forward 5′-GGACCTGACCTGCCGTCTAG-3′, reverse 5′-GAGGAGTGGGTGTCGCTGTT-3′; and U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-CTCGCTTCACGAATTTGCGT-3′.

Transfection of miR-92a mimics and inhibitor. miR-92a mimics, miR-92a inhibitor and their corresponding negative controls (NCs) were purchased from General Biosystems (Anhui) Corporation Ltd. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection according to the manufacturer's instructions. Briefly, the cells were uniformly inoculated into 6-well plates (2×10⁵/well) and cultured at 37°C and 5% CO₂ until the cell confluence reached 30–50% after 24 h. A miRNA storage solution was prepared at a final concentration of 20 µM by dissolving 2.5 µmol freeze-dried miRNA mimics, inhibitor, mimics NC and inhibitor NC in 125 µl deionized water. Subsequently, 10 µl miRNA storage solution was mixed with 240 µl serum-free DMEM; the final concentration of miRNA mimics and inhibitors was 100 nM. The miR-92a mimics, miR-92a inhibitor and their corresponding NCs were then transfected into cells using Lipofectamine 2000 at room temperature for 4–6 h. The subsequent experiments were conducted 48 h post-transfection.

The transfected cells were then cultured in conventional medium for 48–72 h. The sequences were as follows: miR-92a mimics, 5′-UAUUGCACUUGUCCCGGCCUGU-3′; miR-92a inhibitor, 5′-ACAGGCCGGGACAAGUGCAAUA-3′; miR-92a mimics NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-92a inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA-3′.

FOXO3 siRNA (siFOXO3) and NC siRNA (siFOXO3 NC) were acquired from General Biosystems (Anhui) Corporation Ltd. The cells were evenly seeded into 6-well plates (2×10⁵/well) and incubated at 37°C and 5% CO₂ until cell confluence reached 30–50% after 24 h. The cells were cultured with a mixture of siRNA and Lipofectamine 2000 in 100 µl serum-free DMEM, according to the manufacturer's instructions. The medium was replaced after 4–6 h, and the cells were cultured in a 5% CO₂ incubator at 37°C for 48 h. The sequences were as follows: si FOXO3, sense 5′-AAAUAGCUACUUACCUUUGCAGU-3′, antisense 5′-ACUGCAAAGGUAAGUAGCUAUUU-3′; and siFOXO3 NC, sense 5′-UGAAUUGUAAUACGACUCACUAU-3′, antisense 5′-AUAGUGAGUCGUAUUACAAUUCA-3′.

A miRNA storage solution was prepared at a final concentration of 20 µM by dissolving 2.5 µmol freeze-dried miR-92a inhibitor in 125 µl deionized water. A total of 24 h before transfection, the cells were plated in 24-well cell culture plates in complete DMEM (5×10⁴ cells/well). Cells were then transfected with 100 nM miR-92a inhibitor and 100 nM siFOXO3 or siFOXO3 NC for 4–6 h at room temperature using Lipofectamine 2000. Subsequent experiments were performed 72 h post-transfection.

The pmirGLO vector was synthesized by General Biology (Anhui) Co., Ltd. The potential target genes of miR-92a were predicted using StarBase (http://starbase.sysu.edu.cn/) database, which showed that the FOXO3 3′-UTR has a miR-92a-binding site. Logarithmic-phase cells were inoculated into 24-well plates at a density of 2×10⁵ cells/well. Subsequently, the cells were co-transfected with luciferase reporter vectors (FOXO3-WT, 5′-GTAAATTGTTGTGCAATTGTGG-3′; FOXO3-MUT, 5′-GTAAATTGTTGTATGATTGTGGTTA-3′) and miR-92a mimics or NCs using Lipofectamine 2000. Luciferase activity was measured 24 h after transfection using a dual-luciferase reporter kit (Dual Luciferase Reporter Gene Assay Kit; Beyotime Institute of Biotechnology) and luciferase activity was normalized to Renilla luciferase activity.

Each experiment was conducted at least three times and data are presented as the mean ± standard deviation. GraphPad Prism (version 9.0; Dotmatics) was used for statistical analysis. Comparisons between two groups were performed using an unpaired Student's t-test (independent-samples t-test). Comparisons among multiple groups were performed with one-way ANOVA and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Rapamycin, a strong and effective autophagy inducer, is known to stimulate ECs to undergo autophagy when incubated at a concentration of 1,000 nM for 6 h (30). In order to examine the association between miR-92a and autophagy in the EA.hy926 cell line, the cells were exposed to rapamycin at a dose of 1,000 nM for a 6 h to create an autophagy model in EA.hy926 cells. The results of western blotting showed that, compared with in the control group, the expression levels of autophagy-related Beclin1 and the LC3II/I ratio were significantly increased in the rapamycin group (Fig. 1A-C), whereas the expression levels of p62 were decreased (Fig. 1A and D). The results of immunofluorescence assay showed significantly increased fluorescence intensity of LC3B in the rapamycin group compared with that in the control group (Fig. 1E and F). TEM was used to count the number of autophagosomes in EA.hy926 cells in the control and rapamycin-treated groups. The results found that no obvious autophagosomes were present in the cytoplasm of the control group (Fig. 1G), whereas there was an increased number of autophagosomes in the cytoplasm of the rapamycin-treated group (Fig. 1H). These results indicated that rapamycin activated autophagy in EA.hy926 cells, and the rapa-EC.hy926 autophagy model was established successfully.

To assess the association between miR-92a and autophagy in ECs, RT-qPCR was performed to detect the expression levels of miR-92a in the rapa-EA.hy926 cell autophagy model. The findings demonstrated a significant decrease in the expression of miR-92a in the rapamycin group compared with that in the control group (Fig. 1I). These findings indicated that miR-92a was abnormally expressed in the rapa-EA.hy926 autophagy model, suggesting that miR-92a may be involved in autophagy in this model system.

To assess the impact of miR-92a on autophagy in EA.hy926 cells, miR-92a mimics or a miR-92a inhibitor were introduced into rapa-EA.hy926 cells using Lipofectamine. This allowed for the increase or decrease in the levels of miR-92a, hence creating experimental variations in miR-92a expression. The results of RT-qPCR indicated that the miR-92a mimics group exhibited a significant increase in the expression levels of miR-92a, indicating overexpression, compared with those in the mimics NC control group (Fig. 2A). Conversely, the miR-92a inhibitor group showed a significant decrease in the expression level of miR-92a, indicating inhibition. Western blot analysis demonstrated that, compared with those in the control group, the expression levels of Beclin1 and the LC3II/LC3I ratio in the miR-92a mimics group were decreased, and the protein expression levels of p62 were increased (Fig. 2B-E). By contrast, the expression levels of Beclin1 and the LC3II/LC3I ratio were higher in the miR-92a inhibitor group than those in the control group, whereas the protein expression levels of p62 were decreased. In addition, immunofluorescence staining results showed that the fluorescence intensity of LC3B in the miR-92a mimics group was significantly decreased compared with that in the control group, whereas the fluorescence intensity of LC3B in the miR-92a inhibitor group was increased compared with that in the control group (Fig. 2F and G). These findings indicated that the autophagy marker protein LC3B accumulated in the cytoplasm of the miR-92a inhibitor group. Furthermore, as number of autophagosomes were observed by TEM in the cytoplasm of the miR-92a inhibitor group, whereas no autophagosomes were apparent in the miR-92a mimics group, and only a small number of autophagosomes were observed in the miR-92a inhibitor and miR-92a mimic NC groups (Fig. 2H-K). These results suggested that overexpression of miR-92a may inhibit autophagy, whereas inhibition of miR-92a could enhance autophagy in rapa-EA.hy926 cells.

According to reports (31,32), miRNAs control several biological processes of cells by suppressing the transcription or translation of specific target genes at the post-transcriptional stage. Therefore, whether miR-92a is involved in the regulation of autophagy in EA.hy926 cells by acting on downstream target genes was explored. The bioinformatics software StarBase was used to analyze the downstream target genes of miR-92a. The results showed a binding site for miR-92a in the 3′-UTR region of FOXO3 (Fig. 3A); therefore, it was hypothesized that FOXO3 is a target gene for miR-92a. To assess this, FOXO3 wild-type and mutant luciferase reporter plasmids were constructed (Fig. 3B). Notably, luciferase activity in the miR-92a mimics + wild-type FOXO3 plasmid group was significantly lower than that in the miR-92a mimic NC + wild-type FOXO3 plasmid group (Fig. 3D). There was no significant difference in luciferase activity between the miR-92a mimic + mutant FOXO3 plasmid group and the miR-92a mimic NC + mutant FOXO3 plasmid group, which further confirmed that miR-92a directly binds to FOXO3 3′-UTR.

Subsequently, the impact of miR-92a on the control of FOXO3 gene expression was investigated. Western blotting and RT-qPCR showed that FOXO3 mRNA and protein expression levels were inhibited in the miR-92a mimics group compared with those in the control group, whereas FOXO3 mRNA and protein expression levels were enhanced in the miR-92a inhibitor group (Fig. 3C, E and F). In addition, FOXO3 mRNA and protein expression levels were significantly downregulated in the miR-92a mimics group compared with those in the miR-92a inhibitor group. These results indicated that the overexpression or inhibition of miR-92a can affect the expression of FOXO3 mRNA and protein. In summary, these findings suggested that FOXO3 may be a direct target of miR-92a in EA.hy926 cells and that miR-92a directly binds to a specific site on the FOXO3 3′-UTR to regulate its expression.

The present study demonstrated that miR-92a serves a role in controlling autophagy in EA.hy926 cells and that FOXO3 is a gene targeted by miR-92a. In addition, previous research (33) has indicated that FOXO3 has a significant role as an autophagic protein. Therefore, it was hypothesized that miR-92a might regulate the autophagy of EA.hy926 cells through FOXO3. To assess this hypothesis, the present study first investigated whether FOXO3 affected autophagy in EA.hy926 cells. siFOXO3 was transfected into cells using Lipofectamine to silence FOXO3. RT-qPCR and western blot analysis showed that FOXO3 mRNA and protein expression levels were significantly inhibited in the in siFOXO3 group of rapa-EA.hy926 cells compared with in the siFOXO3 NC group (Fig. 4A-C), indicating that FOXO3 siRNA was successfully transfected into cells. Western blotting was used to detect the expression levels of autophagy-related proteins in siFOXO3 cells. The results showed that, compared with in the siFOXO3 NC group, siFOXO3 transfection significantly decreased the expression levels of Beclin1 and the LC3II/I ratio, whereas p62 protein expression levels were increased (Fig. 4A and D-F). In addition, the immunofluorescence intensity of LC3B in siFOXO3 cells was weaker than that in the siFOXO3 NC cells (Fig. 4G and H). Furthermore, TEM revealed that autophagosomes were readily apparent in the siFOXO3 NC group, whereas no autophagosome formation was observed in the siFOXO3 group (Fig. 4I and J). Therefore, it was suggested that FOXO3 gene silencing inhibited autophagy in rapa-EA.hy926 cells.

siFOXO3 partially reversed the promoting effect of miR-92a inhibitors on rapa-EA.hy926 cell autophagy. To evaluate the role of miR-92a in regulating autophagy in EA.hy926 cells via the suppression of FOXO3 expression, the rapa-EA.hy926 cells were transfected with miR-92a inhibitor + siFOXO3 or miR-92a inhibitor + siFOXO3 NC. Western blotting results showed that the expression levels of Beclin1 and the LC3II/I ratio in the miR-92a inhibitor + siFOXO3 group were decreased compared with those in the miR-92a inhibitor + siFOXO3 NC group, whereas the expression levels of p62 showed an opposite trend (Fig. 5A-D). Immunofluorescence results also showed that the fluorescence intensity of LC3B in the miR-92a inhibitor + siFOXO3 group was significantly weaker than that in the miR-92a inhibitor + siFOXO3 NC group (Fig. 5E and F). Similarly, only a few autophagosomes were detected in the cells of the miR-92a inhibitor + siFOXO3 group by TEM (Fig. 5 G and H). In conclusion, the results of siFOXO3 and miR-92a inhibitor co-transfection suggested that siFOXO3 can partially reverse the promoting effect of the miR-92a inhibitor on the autophagic activity of rapa-EA.hy926 cells. This further verified that miR-92a regulates autophagy in EA.hy926 cells by targeting FOXO3.