Suppression of METTL3 expression attenuated matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the extracellular matrix in pelvic organ prolapse.

Pelvic organ prolapse (POP) is a multifactorial disease that is characterized by the descent and dysfunction of the pelvic organs caused by weakness and damage of the supportive structures of the pelvic floor, with a prevalence ranging from 1% to 65% worldwide. [ 1 ] One to 31% of symptomatic patients may suffer from urination and defecation dysfunction, infection, bleeding, and sexual complaints, which impose severe psychological and economic burdens on individuals and society. [ 1 , 2 ] However, owing to the complex and unclear pathogenesis of POP, an effective causative treatment strategy for this disease is still lacking. Current treatments for POP are focused primarily on surgical repair of these structural weaknesses. However, these surgical options are insufficient, and native tissue procedures and synthetic mesh surgery are related to high recurrence and high complication rates, respectively. [ 3 ] Thus, there is a lack of therapies starting from the pathogenesis of POP to provide new effective preventative and therapeutic strategies that can fundamentally repair damaged structural tissue. According to previous studies, fibrosis of the connective tissue in the vaginal wall predominates in POP. [ 4 ] Fibrosis is a common pathological change in various diseases and is characterized by excessive activity of fibroblasts leading to myofibroblast differentiation and abnormal deposition of extracellular matrix (ECM), which in turn leads to tissue fibrosis. [ 5 ] Methyltransferase 3 (METTL3) is the first methyltransferase to be discovered as an m6A “writer”, which plays an important role in modulating many biological processes, such as tumor proliferation and migration, hematological disorders, cell differentiation, and embryonic development. [ 6 – 11 ] In recent years, the silencing of METTL3 has been shown to attenuate tissue fibrosis, including that of myocardial tissue, kidney tissue, and liver tissue. [ 12 – 14 ] However, evidence that matrix stiffness induces fibroblast-to-myofibroblast differentiation and ECM modulation in vaginal fibroblasts is scarce, and the role of METTL3 in mediating the process of matrix stiffness-induced fibroblast-to-myofibroblast differentiation and ECM modulation in vaginal tissue in the development of POP remains unexplored. Our study aimed to investigate the relationships between METTL3 and vaginal fibroblast differentiation and tissue fibrosis and to explore new therapeutic approaches for the prevention and treatment of POP.

This study was supported by the 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 CAMS Innovation Fund for Medical Sciences (No. 2023-I2M-C&T-B-033).

The study was approved by the Peking Union Medical College Hospital (PUMCH) Ethics Committee (No. JS-2240, 25/02/2020). All participants signed written informed consent before tissue collection. Menopausal patients who underwent procedures related to 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 anterior vaginal wall of the non-POP group was obtained from patients who underwent hysterectomy for non-POP benign diseases. Patients who had leiomyoma, endometriosis, or malignant tumors; who had a history of pelvic surgery; or who had undergone hormone treatment were excluded from both groups. Soft and stiff PA gels with varying stiffness were fabricated based on a published protocol and prepared to mimic the cellular environment with different mechanical properties. [ 15 ] Soft and stiff gels were formed from 40% acrylamide stock solution (Servicebio; Wuhan, China) and 2% bis-acrylamide stock solution (Servicebio), respectively, at different final concentrations. A 1/100 total volume of ammonium persulfate (APS; Servicebio) and a 1/1000 total volume of tetramethyl ethylenediamine (TEMED; Servicebio) were added to the gel solutions. A total of 25 μL of the gel mixture was placed on the treated side of a chloro-silanated glass slide with a 25-mm circular amino-silanated coverslip. Once the solution was fully polymerized, 500 μL of sulfo-sufosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)-hexanoate solution (Sigma, St. Louis, MO, USA) per coverslip was added to the gel surface, which was placed in a 365-nm ultraviolet (UV) light source and exposed for 10 min. Finally, the gel surfaces were coated with 0.1 mg/mL rat tail collagen I (Sigma) overnight at 37°C. The gel-coated coverslips were washed once with phosphate-buffered saline (PBS; Servicebio, Wuhan, China) and stored in PBS at 4°C for further experiments. Before plating the cells, the PA hydrogels were sterilized for 30 min under UV light. PA gel mechanical properties were measured using an atomic force microscope (NT-MDT Prima; Bruker Dimension Edge, Moscow, Russia) in contact mode. A minimum of 30 independent measurements were obtained and analyzed for soft and stiff PA gels. The final Young’s modulus was analyzed using JPK software (Berlin, Germany) and determined as the mean ± standard deviation (SD) from all measured locations on PA gel samples. Approximately 1-cm 2 pieces of anterior vaginal wall tissue from the prolapse site were collected during surgery and washed with PBS three times to remove any residual blood clots. The vessels and the epithelial layer of the vaginal wall were removed completely using tissue scissors and a scalpel. The tissue was subsequently cut into small pieces and spread flat in 25-cm 2 cell culture flasks with 3-mm spacing between pieces. The mixture was subsequently cultured at 37°C with 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% Penicillin-Streptomycin-Amphotericin B solution (P/S/B, Servicebio), and 0.1% Mycoplasma Prevention Reagent. After 10–14 days, primary vaginal fibroblasts (passage 0) were grown from the small pieces, and the cells at passages 2–4 were collected and cultured on coverslips covered with different PA gels placed in a 35-mm petri dish or a 6-well plate. Each 25-mm coverslip was transferred to a new 35-mm petri dish or 6-well plate before total cell protein extraction. Proteins were extracted directly from cells cultured on 25-mm coverslips after 72 h. The supernatants were collected carefully, and the protein concentration was detected using a Bradford protein assay (Beyotime, Shanghai, China), after which the proteins were denatured at 70°C for 10 min. Denatured proteins (30 μg each) were subjected to NuPAGE TM 10% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and separated with NuPAGE TM MES SDS Running Buffer (20×, Invitrogen). After 50 min of electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF, Invitrogen) membranes. The membranes were blocked for 30 min and incubated with the following antibodies overnight at 4°C: anti-METTL3 (1:1000; rabbit polyclonal antibody, ABclonal; Wuhan, China); anti-α-smooth muscle actin (α-SMA) (1:1000; rabbit monoclonal antibody, ABclonal); anti-collagen Type I Alpha 1 (COL1A1) (1:800; rabbit polyclonal antibody, ABclonal); anti-collagen Type 3 Alpha 1 (COL3A1) (1:800; rabbit monoclonal antibody, ABclonal); anti-tissue inhibitor of matrix metalloproteinase 1 (TIMP1) (1:1000; rabbit polyclonal antibody, ABclonal); anti-tissue inhibitor of matrix metalloproteinase 2 (TIMP2) (1:1000; Proteintech, rabbit polyclonal antibody, Wuhan, China); anti-glyceraldehyde-phosphate dehydrogenase (GAPDH) (1:1000; rabbit monoclonal antibody, ABclonal). After being washed in tris buffered saline with Triton X-100 (TBST) (Servicebio) three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:4000, Solarbio, Wuhan, China) at room temperature for 2 h. The immunoblots were visualized on a GelView 6000 Plus imaging system (Biolight Biotechnology, Guangzhou, China) with Rapid Step ECL Reagent (Millipore, Schwalbach, Germany). Quantification of the densitometry of immunoblot bands was performed using ImageJ software (version 1.51j8, https://imagej.net/ , National Institutes of Health, USA). Total RNA was extracted from cells cultured on 25-mm coverslips for 48 h using a FastPure Cell Total RNA Isolation Kit V2 (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. The concentration and quality of the RNA were measured with a NanoDrop TM One Spectrophotometer (Thermo Fisher Scientific, CA, USA). For each sample, 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using HiScript III RT SuperMix for qPCR (Vazyme Biotech). RT–qPCR was performed on an Applied Biosystems TM QuantStudio TM 3 (Thermo Fisher Scientific) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech). The reactions were conducted as follows: 30 s at 95°C (hold stage) followed by 40 cycles of 5 s at 95°C and 20 s at 60°C (PCR stage), with a final melting curve stage. GAPDH was used as the internal control for each sample, and the relative gene expression was analyzed using the 2 − ΔΔ CT method. The sequences of the primers were provided in supplementary methods, http://links.lww.com/CM9/C249 . For overexpression, the cells were transfected with a METTL3 overexpression vector plasmid, and subcloned and inserted into the pcDNA3.1 vector by a supplier (Tsingke Biotechnology, Beijing, China). The pcDNA3.1 empty vector (EV) plasmid was used as a negative control provided by the supplier. To knock down METTL3 expression, an RNA interference assay was performed by transfecting cells with small interfering RNA (siRNA) targeting METTL3 messenger RNA (mRNA). Three siRNA sequences for METTL3 were designed (s1: CTACAGATCCTGAGTTAGA; s2: GGAACAATCCATTGTTGAA; s3: CAAGATTGAGTTATTTGGA). A negative control (NC) siRNA was used as a negative control. Various concentrations (10 nmol/L, 20 nmol/L, 50 nmol/L, and 100 nmol/L) of carboxyfluorescein (FAM)-labeled negative control siRNA (FAM-NC-siRNA) were used to determine the optimal transfection concentration. Fluorescence was observed with a Nikon Ti microscope (Ti2-U, Nikon, Tokyo, Japan). To detect the silencing effects of the three METTL3 siRNAs, the cells were divided into control (without siRNA), siRNA1, siRNA2, and siRNA3 groups and evaluated by WB and RT–qPCR to determine the optimal siRNA for use in subsequent experiments. The cells were incubated in an antibiotic-free medium for at least 24 h before transfection. Two microliters of METTL3 siRNA/NC siRNA (20 nmol/L/μL) was added to 250 μL of Opti-MEM ® I Reduced Serum Medium (Gibco, New York, NY, USA) and mixed with 5 μL of Lipofectamine 2000 reagent (Invitrogen) diluted in 250 μL of Opti-MEM ® I Reduced Serum Medium (Gibco), and 4 μg of pcDNA3.1-METTL3/pcDNA3.1 EV (1 μg/μL) was added to 250 μL of Opti-MEM ® I Reduced Serum Medium (Gibco) and mixed with 10 μL of Lipofectamine 2000 reagent (Invitrogen) diluted in 250 μL of Opti-MEM ® I Reduced Serum Medium (Gibco). Both mixtures were kept at room temperature for 20 min. Subsequently, the complex was added to the corresponding cells, which were then incubated for 6 h at 37°C. After a 6-h incubation, the cell medium was replaced with a fresh complete culture medium, and the cells were cultured for another 24–72 h for further experiments. EdU staining was carried out to detect the cellular proliferation capacity via the BeyoClick TM EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime) according to the manufacturer’s instructions. Human vaginal wall fibroblasts cultured on gels with different stiffness were incubated with EdU for 2 h. Cells stained with both red and blue were considered EdU-positive cells. The results of the EdU staining were photographed under an Nikon optical microscope, and the relative nuclear size analysis was determined using ImageJ. The data are presented as the means ± SDs. Two groups were compared using the Student’s t -test. Multiple groups were compared via a one-way analysis of variance (with a post hoc Tukey test). P <0.05 was considered to indicate statistical significance.

Primary fibroblasts were isolated from the vaginal wall tissues of women with POP and cultured for three passages. IF staining was used to identify the phenotype of the primary fibroblasts through the use of specific markers, such as fibroblast-specific protein-1 (FSP-1) and vimentin for fibroblasts, α-SMA for myofibroblasts, cytokeratin and caldesmon for epithelial cells, and desmin for smooth muscle cells. The cultured cells at the third passage were positive for FSP-1, vimentin, and α-SMA but negative for cytokeratin, caldesmon, and desmin, indicating a mixture of fibroblasts and myofibroblasts without epithelial and muscle cells [Supplementary Figure 1, http://links.lww.com/CM9/C249 ]. To investigate the effect of stiffness on vaginal fibroblasts from POP patients, we seeded vaginal fibroblasts on soft and stiff surfaces of variable micro stiffness. Thirty locations on the PA gel samples were measured via atomic force microscopy, and analysis of Young’s modulus revealed a mean stiffness value of 0.20 ± 0.03 kPa for the soft PA gels and 12.12 ± 0.28 kPa for the stiff PA gels, which were in line with the expected values. There is currently no specific study describing the differences in surface topography of PA gels with different stiffness. Our study presented an initial demonstration of the surface topography of PA gels with varying stiffness. Macroscopic differences were not observed between soft and stiff gels. Moreover, the surface morphology of PA gels with different stiffness was observed differently via atomic force microscopy at 450× magnification. The surfaces of the soft PA gels were smooth, whereas the surfaces of the stiff gels were sharp [Figure 1 ]. (A–B) The surface and 3D topography of soft and stiff PA gels were observed via atomic force microscopy. Scan size of the PA gel was 50 µm × 50 µm and the resolution was 1.6 µm. (C) The stiffness of soft and stiff PA gels was measured via atomic force microscopy at multiple locations ( n = 30) and the results were analyzed by Young’s modulus and are presented as the means ± SDs (0.20 ± 0.03 kPa and 12.12 ± 0.28 kPa for soft and stiff gel samples, respectively). PA: Polyacrylamide; SD: Standard deviation. POP is a clinical manifestation of pathological tissue fibrosis caused by excessive activation of vaginal fibroblasts to myofibroblast differentiation combined with variation in the composition of the ECM. We used PA gels with variable stiffness to culture POP vaginal fibroblasts. Increasing microenvironmental stiffness was considered an effective factor related to the progression of POP through excessive activation of vaginal fibroblasts. WB and RT-qPCR revealed increases in α-SMA (a marker of activated-fibroblasts) when vaginal fibroblasts were cultured on stiff PA gels (12 kPa) compared with those in cells cultured on soft PA gels (0.2 kPa) [Figure 2 A]. Moreover, cells cultured on 12 kPa gels presented lower expression of COL1A1, TIMP1, and TIMP2 and a lower COL1A1/COL3A1 ratio than did those cultured on 0.2 kPa [Figures 2 B–E]. It presented higher expression of COL3A1 on 12 kPa gels as well [Supplementary Figure 2, http://links.lww.com/CM9/C249 ]. The results revealed that the number of EdU-labeled proliferating vaginal fibroblasts was significantly greater in the 12 kPa group than in the 0.2 kPa group, indicating that elevated matrix stiffness promoted vaginal fibroblast proliferation [Figure 2 F]. IF indicated the expression of α-SMA increased significantly when vaginal fibroblasts were cultured on stiff PA gels [Figure 2 G]. (A–E) Representative gene expression of vaginal fibroblasts cultured on PA gels with variable stiffness (0.2 kPa vs . 12 kPa). WB analysis of protein expression and RT–qPCR analysis of the mRNA expression of α-SMA (A), COL1A1 (B), TIMP1 (C), TIMP2 (D), COL1A1/COL3A1 (E). n = 5; (F) EdU assays comparing vaginal fibroblast proliferation between 0.2 kPa and 12 kPa. (G) Representative images (IF) showing the effect of matrix stiffness on α-SMA, a marker of fibroblast-to-myofibroblast differentiation. GAPDH was used as an internal control. Scale bar = 100 μm. * P <0.05, † P <0.01, ‡ P <0.001. α-SMA: α-smooth muscle actin; COL1A1: Collagen Type 1 Alpha 1; COL3A1: Collagen Type 3 Alpha 1; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-phosphate dehydrogenase; IF: Immunofluorescence; PA: Polyacrylamide; RT–qPCR: Real-time polymerase chain reaction; TIMP1: Tissue inhibitor of matrix metalloproteinase 1; TIMP2: Tissue inhibitor of matrix metalloproteinase 2; WB: Western blot. METTL3 has been shown to regulate certain types of tissue fibrosis. According to the results of the IHC, IF, and WB analyses, the expression of METTL3 in the vaginal wall tissues of POP patients was significantly greater than that in non-POP patients [Figure 3 A–C]. Using WB and RT−qPCR, we found that METTL3 was enhanced in human POP vaginal fibroblasts by 12 kPa stiffness compared with 0.2 kPa ( P <0.05), thus identifying METTL3 as a mechanosensitive mediator in mechanical stiffness-induced vaginal fibroblasts activation [Figure 3 D]. (A–C) Differential expression of METTL3 in the vaginal wall tissues of non-POP patients and POP patients. (A) IHC; (B) IF; (C) WB. (D) Representative gene expression of vaginal fibroblasts cultured on PA gels with variable stiffness (0.2 kPa vs . 12 kPa). WB analysis of protein expression and RT–qPCR analysis of the mRNA expression of METTL3. * P <0.05, † P <0.01, ‡ P <0.001. DAPI: 4′,6-diamidino-2-phenylindole; IF: Immunofluorescence; IHC: Immunohistochemistry; METTL3: Methyltransferase 3; PA: Polyacrylamide; POP: Pelvic organ prolapse; RT–qPCR: Real-time polymerase chain reaction; WB: Western blot. CTR: Control. We first used siRNA-1 (CTACAGATCCTGAGTTAGA)-targeting METTL3 mRNA, which demonstrated the highest silencing efficiency among three METTL3 siRNAs (siRNA-1, siRNA-2, and siRNA-3) to knock down METTL3 in vaginal fibroblasts on stiff gel according to WB and RT–qPCR [Figure 4 A]. METTL3 siRNA-1 produced approximately 70% reduction in METTL3 mRNA and protein levels [Figure 4 B]. The successful overexpression of METTL3 was confirmed at both the protein and mRNA levels, as revealed by a significant increase [Figure 4 C]. The concentration of 20 nmol/L was determined to be the optimal transfection concentration compared with that of 10 nmol/L, 50 nmol/L, and 100 nmol/L for showing the most living cells with FAM-NC-siRNA [Figure 4 D]. Silencing and overexpression effects of METTL3 siRNA and vector plasmids and the optimal transfection concentration. (A) The silencing efficiency of three METTL3 siRNAs (siRNA-1, siRNA-2, and siRNA-3) according to WB and RT–qPCR. (B) The silencing efficiency of METTL3 siRNA-1 was determined via WB and RT–qPCR. (C) Effects of the METTL3 overexpression vector plasmid on both protein and mRNA levels. (D) The optimal transfection concentration was determined among various concentrations (10 nmol/L, 20 nmol/L, 50 nmol/L, and 100 nmol/L) of carboxyfluorescein FAM-NC-siRNA. It was observed under a microscope at a magnification of 4×. Scale bar = 100 μm. * P <0.05, † P <0.01, ‡ P <0.001. GAPDH: Glyceraldehyde-phosphate dehydrogenase; FAM-NC-siRNA: FAM-labeled negative control siRNA; METTL3: Methyltransferase 3; RT–qPCR: Real-time polymerase chain reaction; siMETTL3: METTL3 siRNA; oe-METTL3: METTL3 overexpression; WB: Western blot. NC: Negative control; CTR: Control. The ability of METTL3 siRNA (siMETTL3) to silence METTL3 expression in vaginal fibroblasts was verified by WB and RT–qPCR [Supplementary Figure 3A and H, http://links.lww.com/CM9/C249 ]. WB, RT–qPCR and IF revealed that METTL3 knockdown inhibited the stiffness-induced increase in α-SMA expression during human vaginal myofibroblast differentiation on a stiff matrix ( P <0.05) [Supplementary Figure 3B, http://links.lww.com/CM9/C249 ]. These findings indicated that siMETTL3 was resistant to the stiff matrix-induced vaginal fibroblast-to-myofibroblast transition. In addition, a significant increase in TIMP1 and TIMP2 and the COL1A1/COL3A1 ratio in the stiff matrix was observed when METTL3 siRNA was used, which indicated that stiffness-induced expression differences were abolished in METTL3-knockdown cells according to both WB [Supplementary Figure 3C-G, http://links.lww.com/CM9/C249 ] and RT–qPCR [Supplementary Figure 3I-N, http://links.lww.com/CM9/C249 ]. Cellular staining for proliferating cells (red-stained cells) carried out via EdU staining was quantified for the detection of the cellular proliferating capacity [Supplementary Figure 3O, http://links.lww.com/CM9/C249 ]. Transfection of siMETTL3 into vaginal fibroblasts on a stiff gel inhibited proliferation compared with untreated cells on a stiff gel. METTL3 expression was increased at both the protein and mRNA levels in vaginal fibroblasts transfected with the METTL3 overexpression plasmid [Supplementary Figure 4A and H, http://links.lww.com/CM9/C249 ]. To determine whether METTL3 upregulation was sufficient to induce vaginal fibroblast-to-myofibroblast differentiation, the cells were transfected with a METTL3 overexpression vector plasmid. WB, RT–qPCR, and IF revealed that METTL3 overexpression significantly promoted the expression of α-SMA in vaginal fibroblasts cultured on a soft matrix ( P <0.05) [Supplementary Figure 4B, http://links.lww.com/CM9/C249 ]. These findings indicated that METTL3 overexpression promoted vaginal fibroblast-to-myofibroblast transition. In addition, significant decreases in TIMP1 and TIMP2 expression and the ratio of COL1A1/COL3A1 in the soft matrix were observed following transfection of the METTL3 overexpression vector plasmid according to both WB [Supplementary Figure 4C-G, http://links.lww.com/CM9/C249 ] and RT–qPCR [Supplementary Figure 4I-N, http://links.lww.com/CM9/C249 ]. To assess whether METTL3 affected the proliferation of vaginal fibroblasts, EdU staining experiments were performed [Supplementary Figure 4O, http://links.lww.com/CM9/C249 ]. The results revealed that the number of EdU-labeled proliferating vaginal fibroblasts was significantly greater in the 0.2 kPa + METTL3-overexpressing group than in the 0.2 kPa group, indicating that METTL3 promoted vaginal fibroblast proliferation. In contrast to the inhibitory effect of METTL3 knockdown on vaginal fibroblasts, METTL3 overexpression promoted collagen expression and fibroblast activation. In all cases, the EV and NC siRNA did not cause any significant changes in METTL3, α-SMA, COL1A1/COL3A1, TIMP1 or TIMP2 expression or vaginal fibroblast proliferation.

Fibrosis predominates in various diseases related to abnormal fibroblast-to-myofibroblast differentiation, and new antifibrotic treatment techniques have been developed. [ 16 , 17 ] Fibroblasts are the most important cellular component of pelvic connective tissue and play a key role in collagen metabolism and the regulation of ECM components. [ 18 – 20 ] Some studies have reported that POP patients have a lower ratio of COL1A1/COL3A1 than non-POP patients, and severe POP patients presented a significant decrease in the level of COL1A1 and an increase in the content of α-SMA and COL3A1 in the vaginal wall tissue compared with non-POP and mild POP patients. [ 4 , 21 ] Our previous study is consistent with this conclusion and revealed that vaginal fibroblasts secrete collagen with greater stiffness in POP patients. [ 22 ] Moreover, the vaginal wall becomes stiffer with the progression of the disease or compared with that of non-POP females. [ 23 – 26 ] In some studies, fibroblasts are able to sense and respond to the mechanical properties of the environment, such as the stiff microenvironment, and some fibroblasts differentiate into myofibroblasts in environments with elevated stiffness. [ 27 – 29 ] According to the current literature, increased matrix stiffness can promote fibroblast-to-myofibroblast differentiation, including that of the heart, lung, liver, and skin. [ 27 , 29 – 32 ] However, the association between fibroblast-to-myofibroblast differentiation and matrix stiffness has been extensively discussed and reported in other tissues, while whether there is a relationship between matrix stiffness and myofibroblast differentiation and to what extent in vaginal tissue are currently unknown. A study examining vaginal fibroblasts cultured on surfaces with varying microstiffness in 6-well plates revealed that the expression level of α-SMA, which is a marker of myofibroblasts, steadily increased with stiffness, indicating a strong correlation between stiffness and fibroblast-to-myofibroblast differentiation. [ 33 ] In our study, we first applied PA gels to vaginal fibroblasts to create more flexible and economical microenvironment stiffness adjustments to analyze the relationship between fibroblast-to-myofibroblast differentiation and stiffness. We found that, compared with those on soft PA gels, vaginal fibroblasts on stiff PA gels presented higher α-SMA expression and a lower ratio of COL1A1/COL3A1 expression. Our study revealed that vaginal fibroblasts underwent myofibroblast differentiation as the microenvironment became stiffer, similar to collagen and other ECM proteins during matrix remodeling. With the progression of POP, the microenvironment of vaginal fibrosis becomes stiffer, which promotes fibroblast-to-myofibroblast differentiation and ECM modulation, and the presence of excessive myofibroblasts over a long period of time causes excessive collagen deposition, and tissue contraction, and promotes tissue fibrosis, which causes the loss of elasticity, resulting in a vicious cycle of eventual dysfunction. Consistent with our study, a study utilizing PA gels with tunable stiffness indicated that matrix stiffness regulates myofibroblast differentiation in pulmonary fibrosis, with the detection of increased α-SMA expression in human lung fibroblasts cultured on stiff PA gels. [ 30 ] Furthermore, we aimed to determine which gene may play a critical role in the progression of matrix stiffness-induced fibroblast-to-myofibroblast differentiation. METTL3 has been shown to participate in regulating various biological processes, such as tumor proliferation and migration, hematological disorders, cell proliferation, and embryonic development. Moreover, several studies have confirmed that the METTL3 gene plays an important role in aggravating tissue fibrosis by promoting the activation of fibroblast-to-myofibroblast differentiation, including in cardiac fibrosis, renal fibrosis, and hepatic fibrosis. A study has demonstrated that upregulation of METTL3 expression enhances the METTL3-m6A pathway to induce hypertrophy in myocardial cells and that inhibition of METTL3 expression is sufficient to prevent hypertrophy in vitro . [ 12 ] Another study applied transforming growth factor-β1 (TGF-β1) to cultured cardiac fibroblasts, which stimulated fibroblast-to-myofibroblast differentiation, and reported that the differentiation of fibroblasts into myofibroblasts was attenuated in response to TGF-β1 stimulation after knockdown of the METTL3 gene in cells. These results suggested that the METTL3 gene plays a key role in regulating fibroblast differentiation. [ 5 ] Similarly, in our study, the stiff microenvironment-induced fibroblast-to-myofibroblast differentiation in vaginal tissue, while silencing METTL3 expression decreased cell proliferation, attenuated fibroblast-to-myofibroblast differentiation, promoted COL1A1 production and deposition, and increased METTL3 expression. These findings suggest that METTL3 plays an important role in regulating the process of matrix stiffness-induced fibroblast-to-myofibroblast differentiation, indicating that suppression of METTL3 expression can alleviate vaginal fibrosis and pelvic dysfunction by attenuating the conversion of fibroblasts to myofibroblasts and affecting ECM modulation. Furthermore, METTL3 participates in various biological processes by mediating m6A modification of mRNAs. However, the downstream targets of METTL3 vary across different tissues. METTL3 has been reported to regulate m6A modification of MALAT1 to promote the progression of renal fibrogenesis by regulating the miR-145/FAK signaling pathway. [ 13 ] ASIC1a was shown to regulate the processing of miR-350 through METTL3-dependent m6A modification, promoting liver fibrosis through the phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/AKT) and extracellular-signal-regulated kinas (ERK) pathways. [ 14 ] POP is characterized by vaginal tissue fibrosis. Therefore, antifibrotic therapy has become a promising strategy for the management of POP with fibrosis. In 2021, Cambridge scholars published a study in Nature on the identification of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) with in vivo activity for the first time and demonstrated that treatment with STM2457 leads to a reduction in acute myeloid leukemia, which is highly clinically important and provides a proof of concept that the targeting of RNA-modifying enzymes represents a promising new avenue for anticancer therapy. [ 34 ] This study also provides ideas for the clinical translation of our research. According to a previous study, the rate of synthetic mesh procedures increased in the past 14 years, our findings provided more evidences that METTL3 might be a new molecular target for the development of alternative approaches for the treatment of POP associated with fibrosis. [ 35 ] In conclusion, PA gels were applied to vaginal fibroblasts to provide different degrees of ECM stiffness in our study, and our findings provide evidence that elevated ECM stiffness induces fibroblast-to-myofibroblast differentiation and ECM modulation in vaginal fibroblasts. More critically, it was proved that METTL3 plays a critical role in regulating the process of matrix stiffness-induced fibroblast-to-myofibroblast differentiation and ECM modulation in vaginal tissue to alleviate vaginal fibrosis during the development of POP. However, the regulatory mechanism of METTL3-mediated methylation of fibrosis-related transcripts on N6-adenosines in the progression of vaginal fibrosis and the downstream target and signaling pathway regulated by METTL3 to promote vaginal fibrosis progression remain uncertain. This topic will be the focus of our future research, and more related reports will be provided in our follow-up studies.

None.