Suppression of METTL3 Expression Attenuates Matrix Stiffness-Induced Vaginal Fibroblast-to-Myofibroblast Differentiation and Abnormal Modulation of the Extracellular Matrix in Pelvic Organ Prolapse

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Suppression of METTL3 Expression Attenuates Matrix Stiffness-Induced Vaginal Fibroblast-to-Myofibroblast Differentiation and Abnormal Modulation of the Extracellular Matrix in Pelvic Organ Prolapse

Authors:
Xiuqi Wang1, Tao Guo1, Xiaogang Li2, Zhao Tian1, Linru Fu1, Zhijing Sun1
1Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, National Clinical Research Center for Obstetric & Gynecologic Diseases, Beijing 100730, China;
2Department of Biobank, National Infrastructures for Translational Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China.

Introduction
Pelvic organ prolapse (POP) is a multifactorial condition characterized by the descent and dysfunction of pelvic organs due to weakened supportive structures of the pelvic floor. It affects 1% to 65% of women globally, with symptoms including urination and defecation dysfunction, infections, bleeding, and sexual complaints. These symptoms impose significant psychological and economic burdens on individuals and society. Current treatments primarily involve surgical repair, but these methods are often insufficient, with high recurrence and complication rates. Therefore, there is a pressing need for therapies that address the underlying pathogenesis of POP to develop effective preventative and therapeutic strategies.

Fibrosis of the connective tissue in the vaginal wall is a predominant feature of POP. Fibrosis is characterized by excessive fibroblast-to-myofibroblast differentiation and abnormal deposition of the extracellular matrix (ECM), leading to tissue stiffness and dysfunction. Methyltransferase 3 (METTL3), a key enzyme in RNA methylation, has been implicated in various biological processes, including tissue fibrosis. However, its role in POP and the mechanisms by which it influences vaginal fibroblast differentiation and ECM modulation remain unclear. This study aimed to investigate the effects of ECM stiffness on vaginal fibroblasts and the role of METTL3 in POP development.

Methods
Patient Selection
The study was approved by the Peking Union Medical College Hospital Ethics Committee. Menopausal patients with anterior vaginal wall prolapse greater than stage II, as assessed by the pelvic organ prolapse quantification (POP-Q) staging method, were included in the POP group. The non-POP group consisted of patients who underwent hysterectomy for non-POP benign diseases. Patients with leiomyoma, endometriosis, malignant tumors, a history of pelvic surgery, or hormone treatment were excluded.

Polyacrylamide Hydrogel Substrate Fabrication
Polyacrylamide (PA) hydrogels with varying stiffness were fabricated to mimic the ECM microenvironment. Soft and stiff gels were created using different concentrations of acrylamide and bis-acrylamide. The gels were coated with rat tail collagen I to simulate the ECM. The mechanical properties of the gels were measured using atomic force microscopy, and the Young’s modulus was calculated.

Cell Culture
Primary vaginal fibroblasts were isolated from the anterior vaginal wall tissues of POP patients and cultured on PA gels with varying stiffness. The cells were characterized using immunofluorescence (IF) staining for specific markers, including fibroblast-specific protein-1 (FSP-1), vimentin, α-smooth muscle actin (α-SMA), cytokeratin, caldesmon, and desmin.

Western Blot and RT-qPCR Analysis
Protein and mRNA expression levels were analyzed using Western blot (WB) and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. Key markers such as METTL3, α-SMA, collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), tissue inhibitor of matrix metalloproteinase 1 (TIMP1), and TIMP2 were assessed.

Knockdown and Overexpression of METTL3
Small interfering RNA (siRNA) targeting METTL3 and an overexpression vector were transfected into vaginal fibroblasts to evaluate the effects of METTL3 silencing and overexpression on fibroblast differentiation and ECM modulation. The optimal transfection concentration was determined using carboxyfluorescein (FAM)-labeled siRNA.

EdU Fluorescence Staining
Cellular proliferation was assessed using 5-ethynyl-2′-deoxyuridine (EdU) staining. The number of EdU-positive cells was quantified to determine the effects of METTL3 modulation on fibroblast proliferation.

Statistical Analysis
Data were presented as means ± standard deviations (SDs). Comparisons between groups were made using the Student’s t-test or one-way analysis of variance (ANOVA) with a post hoc Tukey test. A p-value <0.05 was considered statistically significant.

Results
Identification of Primary Fibroblasts
Primary fibroblasts isolated from POP patients were positive for FSP-1, vimentin, and α-SMA but negative for cytokeratin, caldesmon, and desmin, confirming their fibroblast and myofibroblast phenotype.

Mechanical Properties of PA Gels
The Young’s modulus of the soft and stiff PA gels was 0.20 ± 0.03 kPa and 12.12 ± 0.28 kPa, respectively. Atomic force microscopy revealed smooth surfaces for soft gels and sharp surfaces for stiff gels.

Effects of Matrix Stiffness on Fibroblast Differentiation
Vaginal fibroblasts cultured on stiff PA gels exhibited increased proliferation, higher α-SMA expression, and lower COL1A1, TIMP1, and TIMP2 expression compared to those on soft gels. The COL1A1/COL3A1 ratio was also lower in fibroblasts cultured on stiff gels. These findings indicate that increased matrix stiffness promotes fibroblast-to-myofibroblast differentiation and ECM modulation.

Differential Expression of METTL3
METTL3 expression was significantly higher in vaginal wall tissues and fibroblasts from POP patients compared to non-POP patients. Additionally, METTL3 expression was enhanced in fibroblasts cultured on stiff gels, suggesting its role as a mechanosensitive mediator in fibroblast activation.

Silencing and Overexpression of METTL3
Silencing METTL3 in fibroblasts cultured on stiff gels resulted in decreased proliferation, reduced α-SMA expression, and increased COL1A1/COL3A1 ratio, TIMP1, and TIMP2 expression. Conversely, METTL3 overexpression in fibroblasts cultured on soft gels promoted proliferation, increased α-SMA expression, and decreased COL1A1/COL3A1 ratio, TIMP1, and TIMP2 expression. These results demonstrate that METTL3 plays a critical role in regulating fibroblast differentiation and ECM modulation in response to matrix stiffness.

Discussion
This study provides evidence that elevated ECM stiffness promotes fibroblast-to-myofibroblast differentiation and abnormal ECM modulation in vaginal fibroblasts, contributing to the pathogenesis of POP. METTL3 was identified as a key regulator of these processes, with its expression being mechanosensitive and influencing fibroblast activation and ECM composition.

The findings suggest that targeting METTL3 could be a potential therapeutic strategy for POP. Inhibiting METTL3 expression may attenuate fibroblast-to-myofibroblast differentiation and ECM modulation, thereby alleviating vaginal fibrosis and pelvic dysfunction. Future research should focus on the downstream targets and signaling pathways regulated by METTL3 in vaginal fibroblasts to further elucidate its role in POP.

Conclusion
In conclusion, this study demonstrates that increased ECM stiffness induces fibroblast-to-myofibroblast differentiation and abnormal ECM modulation in vaginal fibroblasts, contributing to the development of POP. METTL3 plays a critical role in regulating these processes, and its suppression may offer a novel therapeutic approach for managing POP. Further research is needed to explore the regulatory mechanisms of METTL3-mediated methylation in vaginal fibrosis and to identify potential downstream targets for therapeutic intervention.

Funding
This study was supported by grants from the National Natural Science Foundation of China (No. 81971366), the Capital Foundation of Medical Development (No. 2024-2-4014), the Beijing Natural Science Foundation-Haidian Original Innovation Joint Fund (No. L232124), and the CAMS Innovation Fund for Medical Sciences (No. 2023-I2M-C&T-B-033).

Conflicts of Interest
None declared.

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DOI:
10.1097/CM9.0000000000003409

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