Demethylase FTO mediates m6A modification of ENST00000619282 to promote apoptosis escape in rheumatoid arthritis and the intervention effect of Xinfeng Capsule

Abstract

Introduction: The pathological mechanisms of rheumatoid arthritis (RA) are closely associated with the apoptosis escape of fibroblast-like synoviocytes (FLS). The m6A modification of long non-coding RNAs (lncRNAs) plays a critical regulatory role in RA pathogenesis. Xinfeng Capsule (XFC), a clinically effective traditional Chinese medicine formulation, has been shown to alleviate RA by inhibiting FLS apoptosis escape. However, its molecular mechanisms remain unclear. This study aimed to elucidate the mechanism by which the demethylase FTO promoted FLS apoptosis escape through the m6A modification of lncRNA ENST00000619282 and to reveal the therapeutic targets of XFC in treating RA by intervening in this m6A-dependent pathway.

Methods: A retrospective analysis was conducted on 1603 RA patients using association rule mining and random walk algorithms to evaluate the efficacy of XFC. The proliferation and apoptosis of co-cultured RA-FLS were assessed using CCK-8, flow cytometry (FCM), and molecular biology techniques. Bioinformatics prediction, MeRIP-qPCR, RIP, and RNA pull-down assays were employed to identify the m6A modification sites of ENST00000619282 and their interactions with FTO/YTHDF1. Additionally, FISH, luciferase reporter assays, and rescue experiments were performed to validate the regulatory role of ENST00000619282 and its sponge-like function in RA-FLS. Clinical samples were analyzed to determine the correlation between FTO/YTHDF1/ENST00000619282/Bax/Bcl-2 and immune-inflammatory markers. Furthermore, the binding affinity of XFC active components to NF-κB was assessed through molecular docking.

Results: Retrospective data mining demonstrated that XFC significantly improved immune-inflammatory markers in RA patients. Mechanistically, FTO reduced the m6A modification level of ENST00000619282, enhancing its stability and promoting YTHDF1-dependent expression, which in turn inhibited PUF60 and activated the NF-κB pathway, ultimately leading to FLS apoptosis escape. XFC downregulated FTO, increased the m6A modification of ENST00000619282, blocked the NF-κB signaling, inhibited RA-FLS proliferation, as well as induced their apoptosis. Clinical validation revealed that FTO/YTHDF1/ENST00000619282/Bax/Bcl-2 was closely associated with immune-inflammatory markers in RA patients. After XFC treatment, FTO, ENST00000619282, and Bcl-2 expressions were decreased, while YTHDF1 and Bax expressions were increased (all P<0.05). Molecular docking confirmed that the active components of XFC (calycosin-7-O-beta-D-glucoside, calycosin, and formononetin) exhibited strong binding affinity to NF-κB p65.

Conclusion: FTO promoted FLS apoptosis escape and RA progression by activating the NF-κB pathway through the m6A-dependent ENST00000619282/YTHDF1 axis. XFC inhibited this pathway by modulating FTO-mediated m6A modification, providing a novel RNA epigenetic regulatory strategy for RA treatment.

Keywords: ENST00000619282; FTO; M6A; Xinfeng Capsule; apoptosis escape; rheumatoid arthritis.

Figure 1

Immune-inflammatory indicators in RA Patients (n = 1603) treated with XFC were evaluated through association rule and random walk analysis. (A) Association rule analysis to examine the correlation between XFC and indicator improvement. (B) Random walk model demonstrating the long-term association between XFC application and indicator improvement.

Figure 2

FTO mediates m6A modification of ENST00000619282. (A) Decreased total m6A level in the model group. (B) Reduced m6A level of ENST00000619282 in the model group. (C) Elevated level of ENST00000619282 in the model group. (D) Protein bands and relative expression levels of FTO, YTHDC1, and YTHDF1. **p < 0.01, ***p < 0.001. Control group: RA-FLS. Model group: co-culture of RA-PBMC and RA-FLS. ns, no significant.

Figure 3

FTO promotes co-cultured RA-FLS proliferation and apoptosis escape by downregulating the m6A modification of ENST00000619282. (A) The effect of FTO knockdown/overexpression on RA-FLS cell proliferation. (B) The effect of FTO knockdown/overexpression on RA-FLS cell apoptosis. (C) The impact of FTO knockdown/overexpression on apoptotic proteins (Bax and Bcl-2). (D) The influence of FTO knockdown/overexpression on the m6A modification of ENST00000619282. (E) The effect of FTO knockdown/overexpression on ENST00000619282 expression. (F) Bioinformatics prediction of two m6A methylation mutation sites on the ENST00000619282 sequence. (G, H) The impact of mutated methylation sites on total m6A and ENST00000619282 m6A levels. (I, J) The effect of mutated methylation sites on total m6A and ENST00000619282 m6A levels when FTO is overexpressed. (K, L) The influence of mutated methylation sites on cell proliferation and apoptosis when FTO is overexpressed. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant.

Figure 4

FTO regulates ENST00000619282 through m6A modification, mediating a YTHDF1-dependent mechanism to promote co-cultured RA-FLS proliferation and apoptosis escape. (A) The impact of YTHDF1 silencing or overexpression on the stability of ENST00000619282. (B, C) The influence of YTHDF1 knockdown/overexpression on the m6A modification and expression levels of ENST00000619282. (D) The effect of overexpressing YTHDF1 and FTO on ENST00000619282. (E, F) The impact of overexpressing YTHDF1 and FTO on cell proliferation and apoptosis. **p < 0.01, ***p < 0.001, ns, no significant.

Figure 5

ENST00000619282 promotes the proliferation and apoptosis escape of co-cultured RA-FLS by inhibiting PUF60. (A) The impact of knockdown/overexpression of ENST00000619282 on the proliferation of RA-FLS cells. (B) The impact of knockdown/overexpression of ENST00000619282 on the apoptosis of RA-FLS cells. (C) The impact of knockdown/overexpression of ENST00000619282 on apoptosis-related proteins (Bax and Bcl-2). (D) RNA pull-down experiment identifying PUF60 as a binding protein of ENST00000619282. (E) RIP experiment demonstrating the interaction between ENST00000619282 and PUF60. (F) FISH experiment confirming the co-localization of ENST00000619282 and PUF60 (DAPI: nucleus, red light: ENST00000619282, green light: PUF60, Merge: combination of DAPI, ENST00000619282, and PUF60). (G) Screening of three silencing sequences for PUF60. (H, I) The impact of knocking down ENST00000619282 and PUF60 on cell proliferation and apoptosis. (J) The impact of knocking down ENST00000619282 and PUF60 on the expression level of PUF60, Bax, Bcl-2, P65, and p-P65 proteins. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

The function of XFC is analyzed. (A–F) The effect of XFC-containing serum on the expression of total m6A, ENST0000619282 m6A, ENST0000619282, FTO, and YTHDF1. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

XFC inhibits co-cultured RA-FLS proliferation and apoptosis escape by regulating the FTO/ENST00000619282/NF-κB Axis. (A) Effects of overexpressing FTO on cell viability with XFC intervention. (B) Effects of overexpressing FTO on cell apoptosis with XFC intervention. (C) Effects of overexpressing FTO on the m6A modification of ENST00000619282 with XFC intervention. (D) Effects of overexpressing FTO on ENST00000619282 expression with XFC intervention. (E) Effects of overexpressing FTO on apoptotic proteins (Bax and Bcl-2) with XFC intervention. (F) Effects of overexpressing FTO and silencing ENST00000619282 on cell viability with XFC intervention. (G) Effects of overexpressing FTO and silencing ENST00000619282 on cell apoptosis with XFC intervention. (H) Effects of overexpressing FTO and silencing ENST00000619282 on the m6A modification of ENST00000619282 with XFC intervention. (I) Effects of overexpressing FTO and silencing ENST00000619282 on ENST00000619282 expression with XFC intervention. (J) Effects of overexpressing FTO and silencing ENST00000619282 on apoptotic proteins (Bax and Bcl-2), p65, p-p65, and PUF60 with XFC intervention. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

Heatmap of correlation analysis between FTO/YTHDF1/ENST00000619282/Bax/Bcl-2 and immune-inflammatory indicators. *P<0.05, **P<0.01, ***P<0.001.

Figure 9

Molecular docking of active components in XFC with the key protein p65 in the NF-κB signaling pathway is conducted. (A) Docking of calycosin-7-O-beta-D-glucoside with P65. (B) Docking of calycosin with P65. (C) Docking of formononetin with P65.

Figure 10

Schematic diagram summarizing the main findings of the present study. Created by Figdraw (ID: AOSRRf5e8f).