[Mechanisms by which Mettl3 regulates pericyte-myofibroblast transdifferentiation through PI3K/AKT signaling pathway].
Zhonghua Xin Xue Guan Bing Za Zhi
; 52(7): 814-826, 2024 Jul 24.
Article
en Zh
| MEDLINE
| ID: mdl-39019831
ABSTRACT
Objective:
To investigate the role and underlying mechanisms of methyltransferase (Mettl) 3 in the process of angiotensin â ¡ (Ang â ¡)-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis.Methods:
C57BL/6J mice were used, in cell experiments, mouse renal pericytes were isolated and cultured using magnetic bead sorting. These pericytes were then induced to transdifferentiate into myofibroblasts with 1×106 mmol/L Ang â ¡, which was the Ang â ¡ group, while pericytes cultured in normal conditions served as the control group. Successful transdifferentiation was verified by immunofluorescence staining, Western blotting, and real-time reverse transcription PCR (RT-qPCR) for α-smooth muscle actin (α-SMA). The levels of m6A modifications and related enzymes (Mettl3, Mettl14), Wilms tumor 1-associated protein (WTAP), fat mass and obesity protein (FTO), ALKBH5, YTHDF1, YTHDF2, YTHDC1, YTHDC2, YTHDC3 were assessed by Dot blot, RT-qPCR and Western blot. Mettl3 expression was inhibited in cells using lentivirus-mediated Mettl3-shRNA transfection, creating sh-Mettl3 and Ang â ¡+sh-Mettl3 groups, while lentivirus empty vector transfection served as the negative control (Ang â ¡+sh-NC group). The impact of Ang â ¡ on pericyte transdifferentiation was observed, and the expression of downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway proteins, including PI3K, AKT, phosphorylated AKT at serine 473 (p-AKT (S473)), and phosphorylated AKT at threonine 308 (p-AKT (T308)), were examined. PI3K gene transcription was inhibited by co-culturing cells with actinomycin D, and the half-life of PI3K mRNA was calculated by measuring residual PI3K mRNA expression over different co-culture time. The reversibility of Mettl3 inhibition on Ang â ¡-induced pericyte-to-myofibroblast transdifferentiation was assessed by adding the AKT activator SC79 to the Ang â ¡+sh-Mettl3 group. In animal experiments, mice were divided into these groups sham group (administered 0.9% sterile saline), Ang â ¡ group (infused with Ang â ¡ solution), sh-Mettl3 group (injected with Mettl3 shRNA lentivirus solution), Ang â ¡+sh-Mettl3 group (infused with Ang â ¡ solution and injected with Mettl3 shRNA lentivirus solution), and Ang â ¡+sh-Mettl3+SC79 group (administered Ang â ¡ solution and Mettl3 shRNA lentivirus, with an additional injection of SC79). Each group consisted of six subject mice. Blood pressure was measured using the tail-cuff method before and after surgery, and serum creatinine, urea, and urinary albumin levels were determined 4 weeks post-surgery. Kidney tissues were collected at 28 days and stained using hematoxylin-eosin (HE) and Masson's trichrome to assess the extent of renal fibrosis.Results:
Primary renal pericytes were successfully obtained by magnetic bead sorting, and intervened with 1×106 mmol/L Ang â ¡ for 48 hours to induce pericyte-to-myofibroblast transdifferentiation. Dot blot results indicated higher m6A modification levels in the Ang â ¡ group compared to the control group (P<0.05). RT-qPCR and Western blot results showed upregulation of Mettl3 mRNA and protein levels in the Ang â ¡ group compared to the control group (both P<0.05). In the Ang â ¡+sh-Mettl3 group, Mettl3 protein expression was lower than that in the Ang â ¡ group, with reduced expression levels of α-SMA, vimentin, desmin, fibroblast agonist protein (FAPa) and type â collagen (all P<0.05). Compared to the control group, PI3K mRNA expression level was elevated in the Ang â ¡ group, along with increased p-AKT (S473) and p-AKT (T308) expressions. In the Ang â ¡+sh-Mettl3 group, PI3K mRNA expression and p-AKT (S473) and p-AKT (T308) levels were decreased (all P<0.05). The half-life of PI3K mRNA was shorter in the Ang â ¡+sh-Mettl3 group than that in the Ang â ¡+sh-NC group (2.34 h vs. 3.42 h). The ameliorative effect of Mettl3 inhibition on Ang â ¡-induced pericyte-to-myofibroblast transdifferentiation was reversible by SC79. Animal experiments showed higher blood pressure, serum creatinine, urea, and 24-hour urinary protein levels, and a larger fibrosis area in the Ang â ¡ group compared to the sham group (all P<0.05). The fibrosis area was smaller in the Ang â ¡+sh-Mettl3 group than that in the Ang â ¡ group (P<0.05), but increased again upon addition of SC79.Conclusion:
Mettl3-mediated RNA m6A epigenetic regulation is involved in Ang â ¡-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis, potentially by affecting PI3K stability and regulating the PI3K/AKT signaling pathway.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Angiotensina II
/
Transducción de Señal
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Fosfatidilinositol 3-Quinasas
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Pericitos
/
Proteínas Proto-Oncogénicas c-akt
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Transdiferenciación Celular
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Miofibroblastos
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Metiltransferasas
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Ratones Endogámicos C57BL
Límite:
Animals
Idioma:
Zh
Revista:
Zhonghua Xin Xue Guan Bing Za Zhi
Año:
2024
Tipo del documento:
Article
País de afiliación:
China