WTAP affects intracranial aneurysm progression by regulating m6A methylation modification

2.1 Establishment of an IA rat model

Sixteen Sprague Dawley rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). All rats were male and weighed between 200 and 250 g; they were fed in a controlled environment with a temperature of 24 ± 2°C, a humidity of 50 ± 5%, and a 12 h light/dark cycle. After acclimatization for 7 days, the animals were randomly divided into the control and IA groups (n = 8 for each group). Throughout the experiment, the animals were allowed to eat and drink freely.

In the IA group, rats were employed to construct a rat IA model using the selective arterial ligation method, as previously reported [26], with some modifications. Briefly, before modeling, the rats were made to fast for 8 h and were deprived of water for 2 h; then, they were generally anesthetized using a combination of sodium pentobarbital intraperitoneal injection (50 mg/kg) and isoflurane inhalation (2%). Following this, the rats underwent ligation of their left common carotid artery and electrocoagulation of the posterior branch of their ventral median renal artery. On the second day following surgery, 1% saline was given to the rats in the IA group instead of drinking water. The control group rats were not ligated and were given normal drinking water.

1. Ethics approval and participation consent: All the animal experiments were conducted in accordance with the relevant guidelines and regulations (National Medical Advisory Committee (NMAC) guidelines and ARRIVE guidelines Declaration of Helsinki) and were approved by the Institutional Animal Care and Use Committee of Changzhou Wujin People’s Hospital (approval no. [2022] 79). Additionally, the animal anesthesia procedures complied with the applicable veterinary guidelines (such as the American Veterinary Medical Association).

2.2 Sample collection and preparation

After 2 months of modeling, all rats were generally anesthetized with a combination of sodium pentobarbital intraperitoneal injection (50 mg/kg, China National Pharmaceutical Corporation) and isoflurane inhalation (2%), the process complied with the applicable veterinary guidelines (such as the American Veterinary Medical Association). Thereafter, the rats were intubated to the renal aorta through the left ventricle via a thoracotomy; blood samples were extracted using an EDTA tube and were preserved. Following this, 30 mL of normal saline was injected through a catheter, while the vena cava was cut open to drain the blood. Three rats were slowly injected through a catheter with 120 mL of 4% paraformaldehyde (China National Pharmaceutical Group Corporation)/0.1 M phosphoric acid buffer (pH 7.4, China National Pharmaceutical Group Corporation) for fixation; the other rats remained without this treatment. Thereafter, cervical dislocation was applied to kill the rats, and their brains were removed via craniotomy. The circle of Willis of each brain was carefully separated under a 10–16× surgical microscope. Among them, the three fixed circle of Willis samples were subjected to hematoxylin and eosin (HE) staining; the remaining five and blood samples were used to isolate the total RNA using the Trizol method.

2.3 Measurement of m6A methylation levels

The blood and circle of Willis samples in each group were employed for total RNA isolation using an RNAiso Plus kit (Trizol, Takara) in line with the manufacturer’s instructions. Thereafter, the isolated RNA was used for qualitative and quantitative detections, with a microplate reader (Thermo Scientific, Wilmington, DE, USA). Following this, the RNA m6A methylation levels in the blood and circle of Willis samples of each group were examined using an EpiQuik™ m6A RNA Methylation Quantification Kit (Colorimetric, Epigentek Group Inc., USA), according to the manufacturer’s protocols.

2.4 Quantitative reverse transcription PCR (RT-qPCR)

The aforementioned isolated RNA was reverse transcribed into cDNA using a commercial cDNA synthesis kit (Takara), according to the manufacturer’s instructions. The total RT-qPCR volume was 20 μL, and the displayed RT-qPCR reaction was as follows: 50°C for 3 min, 95°C for 3 min, a total of 40 cycles at 95°C for 10 s and 60°C for 30 s, followed by 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The sequences of all the designed primers are shown in Table 1; GAPDH was a housekeeping gene. The relative mRNA levels of the related genes were obtained using the 2^−△△Ct method [29].

2.5 Cell culture and cell transfection

The rBMVECs were acquired from Procell Life Science & Technology Co., Ltd (Wuhan, China) and were cultured in Dulbecco’s modification of Eagle’s medium (Gibco, USA), which contained 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco). The cells were stored in an incubator that was supplemented with 5% CO₂ at 37°C and passed when the cells grew to 80–90% confluence.

The pcDNA3.1(+)-WTAP plasmid (oe-WTAP) and pcDNA3.1(+) plasmid (oe-NC) were prepared and synthesized by Yanzai Biotechnology (Shanghai) Co. Ltd; the cell transfection methods were the same as those reported previously [30]. The rBMVECs in good condition were harvested and inoculated into a six-well plate (6 × 10⁵ cells/well). Following overnight incubation, the serum-free medium was replaced with the original medium, and either 2.5 μg of pcDNA3.1(+)-WTAP plasmids or pcDNA3.1(+) plasmids were transfected into the rBMVECs using Lipofectamine 200 (Thermo Fisher Scientific). After 6 h of transfection, the complete medium was added and cultured for another 48 h. The RNA and proteins were extracted from the differently treated cells, whereas the WTAP mRNA and protein expressions were assessed using RT-qPCR and western blotting, to estimate the cell transfection efficiency. The WTAP sequences are displayed in Table 1.

2.6 Cell viability and apoptosis assays

The cells in good condition were collected and inoculated into a 96-well plate (5 × 10³ cells/well). Following overnight culture, the rBMVECs were randomly divided into either a control, TNF-α, or TNF-α + oe-WTAP group. First, the rBMVECs in the TNF-α and TNF-α + oe-WTAP groups were induced by TNF-α (2 ng/mL) for 48 h, and thereafter, the rBMVECs in the TNF-α + oe-WTAP group were transfected with the pcDNA3.1(+)-WTAP plasmid. The cells in the control group remained without treatment. Following 24, 48, and 72 h of culture, 10 μL of the cell counting kit-8 (Beyotime Biotechnology) reagent was supplemented to each well, and the mixture was incubated for another 2 h. Finally, a microplate reader was applied to test the absorbance at 450 nm.

The rBMVECs from the different treatments were acquired to determine cell apoptosis, using an Annexin V-FITC apoptosis assay kit (Beyotime Biotechnology). Briefly, the cells were resuspended with 1× Binding Buffer (100 μL), and thereafter 5 μL of Annexin V-FITC with 5 μL of propidium iodide was added. Following 15 min of incubation in the dark, at 25°C, 400 μL of 1× Binding Buffer was added; thereafter a flow cytometer was employed to acquire the cells, and the total cell apoptosis rate (early apoptosis rate + late apoptosis rate) was calculated.

2.7 Western blot

The rBMVECs were lysed using a RIPA lysis buffer (Beyotime Biotechnology) for total protein extraction, and a BAC assay kit (Boster, Wuhan, China) was employed to determine their concentrations. Subsequently, the protein samples were segregated using 10% SDS-PAGE, transferred to PVDF membranes, and then sealed using 5% skim milk. Following 2 h of incubation, the primary antibodies, including the anti-WTAP antibody (1:5,000, Proteintech Group, Inc.), anti-cleaved caspase3 (1:1,000, Proteintech Group, Inc.), and anti-GAPDH antibody (1:1,000, Proteintech Group, Inc.), were added to the membranes. After being incubated further, at 4°C overnight, the secondary antibody (goat anti-rabbit/mouse IgG-HRP, 1:5,000, Jackson ImmunoResearch) was added. Thereafter, following incubation at 37°C for 2 h, the protein bands were visualized using an ECL assay kit (Beyotime Biotechnology), and Image-Pro Plus (version 6.0, Media Cybernetics Imaging Technologies Inc., USA) was employed to quantify these protein bands.

2.8 Statistical analysis

All the data were expressed as the mean ± standard deviation, and each experiment was done in triplicate. The statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, Inc.). For differential analysis between two groups, Student’s t-test was performed; a one-way ANOVA followed by Tukey’s post hoc test was conducted for the multiple group analyses. The threshold for the statistical difference was set as P < 0.05.

3.1 Histopathological changes and the m6A methylation level in an IA rat model

HE staining showed that the circle of Willis in the control rats were intact; whereas, in the IA rats, the inner elastic layer of the circle of Willis was damaged (Figure 1a). This indicated that the rat IA model was successfully established. Thereafter, the m6A methylation levels in the blood and circle of Willis samples of the control and IA rats were determined. It was clear that the m6A methylation levels in the blood and circle of Willis samples of the IA rats were both significantly decreased (P < 0.05) in comparison with the control rats (Figure 1b and c).

Figure 1

Histopathological changes of the circle of Willis and m6A methylation levels in an IA rat model. (a) Histopathological changes in the circle of Willis samples of the IA rats using HE staining. The m6A methylation levels in the blood samples (b) and circle of Willis samples (c) of the IA rats. *P < 0.05, as compared to the corresponding samples from control rats.

Thereafter, we assessed the expression level of the methylation-related genes (METTL3, METTL14, and WTAP) and demethylation-related genes (ALKBH1, VIRMA, and FTO) in the blood and circle of Willis samples of the different rats. In the blood samples, there were no significant differences (P > 0.05) in the relative expressions of METTL3, METTL14, VIRMA, and FTO, between the control and IA rats (Figure 2a). Relative to the control rats, WTAP expression was evidently downregulated (P < 0.05) whereas ALKBH1 expression was markedly upregulated in the IA rats (P < 0.05, Figure 2a). Furthermore, in the circle of Willis samples, expressions of METTL3, METTL14, and WTAP were significantly downregulated in the IA rats, as compared to the samples from control rats (P < 0.05, Figure 2b). For the demethylation-related genes, it was observed that the ALKBH1 expression was significantly elevated, whereas the VIRMA expression was evidently downregulated in the IA rats, as compared to the corresponding samples from the control rats (P < 0.05); the expression of FTO showed no obvious differences between the control and IA rats (P > 0.05, Figure 2b). All outcomes suggested that m6A methylation can be reduced in the IA rats.

Figure 2

Relative mRNA expressions of the methylation-related genes (METTL3, METTL14, and WTAP) and demethylation-related genes (ALKBH1, VIRMA, and FTO) in the rats. Expressions of VIRMA, METTL3, FTO, METTL14, ALKBH1, and WTAP in the blood samples (a) or the circle of Willis samples (b) of the rats. *P < 0.05, as compared to the corresponding samples from control rats.

3.2 Methylation-related gene expressions in the TNF-α-induced rBMVECs

TNF-α was applied to stimulate the rBMVECs in order to mimic the IA cell model in vitro [31]; thereafter, the TNF-α and methylation-related gene levels were determined. It was noted that the TNF-α mRNA expression in the control group and the TNF-α-induced rBMVECs were 1.00 ± 0.048 and 5.21 ± 0.155, respectively; this showed a significantly higher level of TNF-α in the TNF-α-induced rBMVECs than that of the control rBMVECs (Figure 3a). This indicated that the TNF-α-induced rBMVECs mimicked the IA model in vitro and could be used for further study.

Figure 3

Expression of the methylation/demethylation-related genes in the TNF-α-induced rBMVECs. (a)Level of TNF-α in the cells treated with TNF-α. (b) Expressions of ALKBH1, METTL3, VIRMA, METTL14, WTAP, and FTO in the TNF-α-induced rBMVECs. *P < 0.05, as compared to the samples from the control cells.

Thereafter, it was found that TNF-α did not significantly influence METTL3 and METTL14 expression levels compared to those of the control rBMVECs (P > 0.05, Figure 3b). In comparison to the control rBMVECs, TNF-α induction evidently downregulated WTAP expression (P < 0.05) and upregulated ALKBH1 expression (P < 0.05, Figure 3b). Moreover, VIRMA expression was significantly lower in the TNF-α-induced rBMVECs than the control rBMVECs (P < 0.05) but FTO expression was markedly upregulated in the TNF-α-induced rBMVECs (P < 0.05, Figure 3b). Due to the consistency and significance of RT-qPCR results in the blood, circle of Willis, and TNF-α-induced rBMVECs samples, WTAP was selected as the focus of our subsequent experiments.

3.3 Cell transfection efficiency

The cell transfection efficiency was assessed by measuring the mRNA and protein expressions of WTAP. It was clear that the WTAP relative mRNA expression in the blank and oe-NC groups were 1.00 ± 0.075 and 1.19 ± 0.085, respectively; no significant difference was observed between the two groups (P > 0.05, Figure 4a). Nevertheless, when the rBMVECs were transfected with the pcDNA3.1(+)-WTAP plasmid, the relative expression of WTAP reached 707.92 ± 52.634, which was 700 times the value observed in the control group and significantly elevated compared to that of the blank cells (P < 0.05, Figure 4a). Additionally, the western blot analysis also showed that the WTAP protein expression in the blank and oe-NC cells exhibited no significant differences (P > 0.05), whereas following the transfection of the pcDNA3.1(+)-WTAP plasmid, the WTAP protein expression was evidently increased (P < 0.05, Figure 4b). All the findings indicated that rBMVECs with WTAP overexpression were successfully constructed and could be used for further study.

Figure 4

Cell transfection efficiency in the transfected rBMVECs. RT-qPCR and western blotting were employed to measure the WTAP mRNA (a) and protein (b) expression levels in the transfected rBMVECs. *P < 0.05, as compared to those of the blank cells.

3.4 Roles of WTAP overexpression in the growth of TNF-α-induced rBMVECs

To further investigate the roles of WTAP on the growth of the TNF-α-induced rBMVECs, TNF-α was applied to treat rBMVECs. After 24 h of culture, the viability in the control cells, TNF-α-induced rBMVECs, and TNF-α-induced rBMVECs with WTAP overexpression showed no significant difference (P > 0.05, Figure 5a). However, after being incubated for 48 and 72 h the cell viability was significantly decreased (P < 0.05) in the TNF-α-induced rBMVECs compared to those in the control cells. Cell viability was significantly increased (P < 0.05) upon WTAP overexpression compared to that of TNF-α-induced cells, and recovered to a similar degree in control cells (P > 0.05, Figure 5a). Therefore, for cell apoptosis detection, the rBMVECs that were cultured for 48 h were selected.

Figure 5

Roles of WTAP overexpression in the growth of the TNF-α-induced rBMVECs. (a) Cell viability of the rBMVECs, with different treatments, following 24, 48, and 72 h of culture. (b) Apoptosis of the rBMVECs, with different treatments, determined using flow cytometry. (c) Cleaved-caspase3 protein expression detected using western blotting. (d) WTAP protein expression detected using western blotting. *P < 0.05, as compared to the samples from the control cells; ^# P < 0.05, as compared to those from the TNF-α cells.

Cell apoptosis of the rBMVECs induced by TNF-α was examined, and the acquired representative images of the different groups are shown in Figure 5b. The total cell apoptosis rates in the control, TNF-α, and TNF-α + oe-WTAP groups were 5.17 ± 0.082, 19.79 ± 5.52, and 11.92 ± 0.33%, respectively. These results indicated that TNF-α induction significantly increased (P < 0.05) the cell apoptosis of the rBMVECs compared to the results of the control cells. Cell apoptosis in the TNF-α + oe-WTAP rBMVECs was evidently reduced (P < 0.05, Figure 5b), compared to that in the rBMVECs induced by TNF-α. These results revealed that TNF-α can enhance the apoptosis of rBMVECs, whereas WTAP overexpression can inhibit the apoptosis caused by TNF-α.

Finally, the protein expressions of cleaved-caspase3 and WTAP in the cells with different treatments were detected. It was found that the tendency of cleaved-caspase3 protein expression in the different cells was similar to that of the cell apoptosis in the different groups (Figure 5c). WTAP protein expression was evidently downregulated (P < 0.05) in the TNF-α-induced rBMVECs compared to that in the control cells. However, relative to TNF-α-induced rBMVECs, WTAP protein expression was significantly upregulated (P < 0.05) in the TNF-α-induced rBMVECs with WTAP overexpression (Figure 5d).