Unveiling novel regulatory mechanisms of miR-5195-3p in pelvic organ prolapse pathogenesis

Introduction

Pelvic organ prolapse (POP) is a prevalent condition among women, characterized by the descent or protrusion of the vaginal wall, uterus, or top of the vagina into the vaginal canal. This condition leads to symptoms like pelvic pressure, vaginal bulging, urinary difficulties, incomplete bowel movements, and sexual dysfunction [1–3]. Although a significant proportion of women (around 41%–50%) exhibit varying degrees of prolapse, the majority of them (approximately 97%) do not experience significant symptoms [4–6]. The prevalence of POP is higher in older women and increases with age, peaking between 60 and 69 years [7–9]. It is projected that by 2050, the number of women affected by POP will rise by 46%, with an estimated 4.9 million cases [10–13]. Various factors contribute to the occurrence of POP, including pregnancy, childbirth, aging, menopause, overweight or obesity, chronic coughing, chronic constipation, repetitive heavy lifting, and a family history of pelvic organ support disorders [14–16].

The management of POP involves non-surgical and surgical interventions [17–19]. Non-surgical approaches such as uterine support and pelvic floor muscle exercises are practical for mild POP but offer limited benefit for severe cases [19, 20]. Although surgery is a standard treatment option for severe POP, it is associated with a high recurrence rate [21]. Therefore, there is a need to identify effective treatment methods to reduce the incidence and postoperative recurrence of POP. However, the exact underlying mechanisms of POP have yet to be fully elucidated, and further research is required to understand its etiology.

In examining the pathological mechanisms of POP, abnormalities in the composition and structure of the extracellular matrix (ECM) are considered crucial factors [7, 19, 22–25]. The ECM is a three-dimensional molecular network in the pelvic connective tissue vital in maintaining pelvic support structures [26, 27]. Previous studies have demonstrated that disruptions in the ECM may weaken these support structures, thus contributing to the development of POP [7, 27–30]. The ECM consists of various proteins, such as collagen and elastin, as well as polysaccharides and proteoglycans, which interact to produce biological activity [31–33]. The homeostasis of the ECM relies on the balance between the metabolic processes of fibrous connective proteins (e.g., collagen and elastin) and the regulatory role of fibroblasts [34–36]. However, in prolapsed tissue, the imbalance in elastin and collagen metabolism disrupts the synthesis and degradation equilibrium of the ECM [37–40].

MicroRNA (miRNA), a class of small RNAs approximately 22 nucleotides in length, plays a crucial role in ECM metabolism by binding to the 3′ untranslated region of messenger RNA (mRNA) and inhibiting its translation or degradation [41–43]. Previously identified in small RNA sequencing of human acute lymphoblastic leukemia in 2011, miR-5195-3p has been reported to have an oncogenic role in tumor development [44–47]. However, its regulatory function in ECM metabolism and pelvic POP progression remains unknown.

The lysyl oxidase (LOX) protein family, a type of secreted copper-dependent amine oxidase, plays a significant role in the biomechanical changes of collagen and elastin [48–51]. LOX is involved in critical ECM composition and stability stages, influencing cell fate, adhesion, growth, differentiation, migration, and the development of various disease states [52, 53]. Studies have shown decreased LOX protein expression in the vaginal tissues of POP patients [54, 55], suggesting that LOX downregulation may contribute to pelvic tissue assembly defects and promote POP development [56, 57]. Additionally, transforming growth factor β1 (TGF-β1), a critical factor in ECM production, is downregulated in the uterosacral ligaments of POP patients. It is accompanied by a decrease in collagen fibers and elastin, as well as an increase in matrix metalloproteinase (MMP)-2/9 activity, which negatively correlates with the degree of prolapse [58–62].

In summary, the regulation of ECM metabolism plays a crucial role in developing POP, with miRNAs playing a significant role in this regulation. Therefore, this study aims to investigate the function and molecular mechanisms of miR-5195-3p in the ECM metabolism of POP patients, providing novel insights and potential targets for POP treatment.

Materials and methods

Cell culture

The mouse fibroblast cell line (3 T3-L1) (CL-006, Wuhan PriCells Biotechnology Co., Ltd., Wuhan, China) was cultured in Dulbecco's Modified Eagle Medium (DMEM) (11965092, Gibco, USA) containing 10% Fetal Bovine Serum (FBS) (TMS-016, Sigma Aldrich, USA), 10 μg/ml streptomycin (85886, Sigma Aldrich, USA), and 100 U/ml penicillin (9073-60-3, Sigma Aldrich, USA). The cells were maintained in a humidified incubator at 37°C with 5% CO₂ (Heracell Vios 160i CR CO2 Incubator, 51033770, Thermo Scientific, Germany). Subculturing was performed when the cell growth reached 80%–90% confluence.

Animal model

Postpartum Sprague-Dawle (SD) rats [63], female, weighing 205–235 g, were selected. All animal studies adhered to the Guide for the Care and Use of Laboratory Animals. The rats were treated with or without miR-5195-3p inhibitor. After 5 days of miR-5195-3p inhibitor administration, the anterior vaginal wall was harvested for further analysis.

Gene screening and analysis of differential expression genes

This study aimed to assess the gene expression patterns associated with POP by analyzing the GSE12852 dataset retrieved from the Gene Expression Omnibus (GEO) public database. The dataset comprised gene expression information from eight POP patients and nine control group females without POP. All participants underwent hysterectomy for benign diseases, and uterosacral and cardinal ligaments were collected during surgery. Several bioinformatics tools were employed to preprocess the data, including background noise correction, data normalization, and gene annotation. For the screening of differentially expressed genes (DEGs), we set strict thresholds: |log2(Fold Change)| > 1 and P < 0.05 [64]. It was done to ensure a comprehensive identification of DEGs, selecting genes with both biological significance and statistical relevance. The aim is to capture a broader range of genes potentially related to the pathogenesis of POP, thereby gaining a more comprehensive understanding of the molecular mechanisms involved. Additionally, we integrated the GeneCards and Comparative Toxicogenomics Databas (CTD) databases, which provide comprehensive gene annotation and evidence on gene–disease associations, respectively. Through a Venn diagram analysis, we cross-referenced data from different sources and eventually identified critical genes significantly associated with POP.

Gene ontology functional classification and enrichment analysis with heatmap visualization

We conducted gene ontology (GO) analysis on the DEGs of interest to uncover their functional classification and enriched pathways in biological processes (BPs), cellular components (CCs), and molecular functions (MFs). This analysis used a significance level of p.adjust <0.01 and various bioinformatics tools. We identified POP’s prominent biological functions and pathways through functional enrichment analysis. Furthermore, we utilized heatmap visualization techniques to analyze the enriched terms, revealing the distribution of genes in different categories and emphasizing the significance of genes like COL3A1 and LOX in multiple BP.

Construction of protein–protein interaction network

Using the STRING database, we constructed a protein–protein interaction (PPI) network to investigate potential connections between critical genes and their encoded proteins. Our analysis revealed that the interaction between LOX and COL3A1 is particularly significant in understanding the pathogenic mechanisms of POP. By examining this PPI network, we gained insights into how proteins collectively interact and how these interactions affect the stability and function of the extracellular matrix. This study provides valuable new insights into the pathogenic mechanisms underlying POP.

Methods for collection and preservation of vaginal wall tissue in patients after total hysterectomy

This study was approved by the Ethics Committee of Shengjing Hospital, China Medical University (2022PS515K). A total of 30 patients who underwent total hysterectomy were enrolled in the study. Among them, 20 patients underwent surgery for pelvic POP, while the other 10 patients underwent total hysterectomy for other benign conditions. Based on the POP-Q score, the 10 patients with POP were further divided into two groups: mild POP (POP I–II) and severe POP (POP III–IV). Following the surgery, tissue samples were collected from the area near the midline of the anterior vaginal wall. The collected samples were divided into two parts: one was fixed in 4% formalin for further analysis, and the other was preserved by freezing in liquid nitrogen for future studies.

Real-time reverse transcription PCR

Initially, total RNA was extracted from frozen vaginal wall tissue or primary cultured cells utilizing the Trizol method (1). Subsequently, reverse transcription was carried out following the instructions provided in the kit. Real-time reverse transcription PCR (qRT-PCR) analysis was then conducted employing the qPCR master mix kit as per the manufacturer’s guidelines. The relative expression levels of miRNAs were determined using the 2^-ΔΔCT method, with U6 or glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH) serving as an internal reference.

The primer sequences used for the PCR reactions were as follows: -miR-5195-3p: Forward primer 5′- AACCCCTAAGGCAACTGGAT −3′, Reverse primer 5′- GAACATGTCTGCGTATCTC -3′. -U6: Forward primer 5′- AAAGCAAATCATCGGACGACC -3′, Reverse primer 5′- GTACAACACATTGTTTCCTCGGA -3′. -LOX: Forward primer 5′- AGGCCACAAAGCAAGTTTCTG −3′, Reverse primer 5′- AACAGCCAGGACTCAATCCCT -3′. -GAPDH: Forward primer 5′- TGTGGGCATCAATGGATTTGG −3′, Reverse primer 5′- ACACCATGTATTCCGGGTCAAT −3′.

Fluorescence in situ hybridization

The vaginal wall tissue was fixed in 4% formalin for 48 h. It was then dehydrated in gradient alcohol and embedded in paraffin. The tissue was sliced into 5 μm thick sections using a microtome and dried. The sections were processed following the instructions of a commercial reagent kit.

To prepare the paraffin sections for subsequent analysis, they were baked in an oven at 60°C for 4 h. The slices were then dewaxed in xylene and dehydrated in gradient alcohol. Proteinase K was applied at 37°C for 20 min, followed by three washes with 2 × Saline Sodium Citrate (SSC) solution at room temperature.

After the gradient alcohol dehydration, the sections were denatured in a deionized formamide solution at 78°C for 8 min. They were then dehydrated again in gradient alcohol and denatured in a hybridization buffer at 73°C for 5 min. Next, the denatured probe mixture was incubated at 37°C overnight, followed by a 15-min wash with deionized formamide solution.

The slices were washed twice, each involving a 10-min incubation in 2 × SSC solution. Finally, the slices were incubated with 4′,6-Diamidino-2-Phenylindol (DAPI), treated with an anti-fluorescence quencher, sealed, and observed using a fluorescent microscope.

Immunohistochemistry

First, tissue samples embedded in paraffin were sectioned and baked at 60°C for 4 h. Subsequently, deparaffinization was conducted using xylene, followed by hydration in a graded ethanol series. After that, the sections were washed thrice in Phosphate-Buffered Saline (PBS) and subjected to antigen retrieval by heating at 120°C for 90 s. They were then cooled to room temperature and washed thrice with PBS. To inhibit endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide at room temperature for 15 min, followed by three washes with PBS. Subsequently, they were incubated at room temperature for 20 min.

Next, the sections were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal antibodies against collagen I (COL I) (1:1000), tissue inhibitor of metalloproteinase-1 (TIMP1) (1:500), ECM2 (1:200), MMP-1 (1:400), LOX (1:100), MMP2 (1:200), MMP9 (1:200), and TGF-β (1:400). Following the primary antibody incubation, the sections were washed three times with PBS, incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibodies at room temperature for 1 h, and washed three more times with PBS. Then, they were incubated with streptavidin-horseradish peroxidase at room temperature for 1 h. After the incubation, the sections were incubated for 30 min at room temperature and washed twice with PBS.

For staining, DAB was applied to the sections for 1–3 min, and then, the sections were rinsed with tap water for 10 min. Hematoxylin staining was performed for 2 min. The sections were briefly differentiated with hydrochloric acid in ethanol, rinsed with tap water for 10 min, dehydrated with graded ethanol, cleared with xylene, and mounted with a neutral mounting medium. Finally, the sections were observed and photographed under a light microscope.

Western blot

To analyze the proteins present in frozen vaginal wall tissues or primary cultured cells, begin by extracting the proteins using Radio-Immuno Precipitation Assay (RIPA) lysis buffer. Determine the protein concentration of the extracted samples using the Bicinchoninic Acid (BCA) method. Adjust the overall protein concentration to 2 μg/μl. Then, load 20 μg of the total protein onto an SDS-polyacrylamide gel for electrophoresis.

Once the proteins have been separated on the gel, transfer them onto a Polyvinylidene Fluorid (PVDF) membrane. To prepare the membrane for antibody detection, incubate it with 5% skim milk at room temperature for 1 h. After that, incubate the membrane with specific rabbit polyclonal antibodies targeting COL I (1:1000), TIMP1 (1:1000), ECM2 (1:200), MMP-1 (1:500), GAPDH (1:5000), LOX (1:500), TGF-β1 (1:1000), phosphorylated Smad2 (p-Smad2, 1:1000), phosphorylated Smad3 (p-Smad3, 1:2000), Smad2 (1:1000), and Smad3 (1:500) overnight at 4°C.

To remove any unbound antibodies, wash the membrane three times with Tris-Buffered Saline with Tween (TBST) for 10 min during each wash. Then, incubate the membrane with a goat anti-rabbit secondary antibody at room temperature for 1 h, followed by three additional washes with TBST for 10 min each. Finally, quantify the relative protein expression levels using GAPDH as the internal reference.

Primary culture and identification of vaginal wall fibroblasts

First, fresh vaginal wall tissue is rinsed with PBS containing penicillin to remove the muscle layer and epithelial tissue and then cultured in a serum-free DMEM. The tissue is cut into small 1 mm³ pieces and placed in cell culture flasks containing DMEM with 1% type I collagenase. It is then incubated at 37°C with shaking every 5 min for 2 h. The supernatant is collected by passing through a 200-mesh cell strainer. The cells are resuspended in DMEM and transferred to 25 cm² cell culture flasks for further incubation at 37°C in a cell culture incubator with 5% CO₂. The cells are suspended on glass slides in a six-well plate and incubated overnight at 37°C and 5% CO₂ after digestion with trypsin. Then, the cells are incubated overnight at 4°C with rabbit polyclonal anti-vimentin antibody (1:50). After three washes, the cells are incubated with fluorescent secondary antibody at room temperature under light avoidance conditions for 1 h. After three additional washes, the cells are incubated with DAPI at room temperature under light avoidance conditions for 30 min. After three more washes with PBS, the samples are sealed with an anti-fluorescence quenching agent. Finally, the samples are observed and photographed using a fluorescence microscope.

Cell transfection

Before conducting cell transfection, logarithmic phase cells were selected and evenly distributed into a 6-well plate after trypsin digestion. Approximately 1 × 10⁶ cells were seeded in each well and cultured until reaching a density of 70%. Following the instructions, the transfection was performed using Lipofectamine 3000 transfection reagent. The transfection involved using the miR-5195-3p inhibitor, LOX siRNA, TGF-β overexpression plasmid, and corresponding negative controls. After transfection, the cells were cultured for 48 h before being collected for subsequent experiments. The sequences used for transfection were as follows: Inhibitor NC (negative control) - 5’-CAGUACUUUUGUGUAGUACAA-3′; miR-5195-3p inhibitor - 5’-AGCCCCCUCAGAGAACUGGAU-3′; si-NC (negative control) - 5’-CACUGAUUUCAAAUGGUGCUAUU-3′; si-LOX - 5’-GUAAUUACAGAAUUGAAACACUGUGUU-3′.

Cell immunofluorescence

After cell transfection, the cells were cultured overnight at 37°C in a CO₂ incubator. Subsequently, the cells were fixed with 4% formaldehyde and permeabilized using 0.5% Triton X-100. To prevent nonspecific binding, the cells were then blocked with donkey serum. Following this, the cells were incubated overnight at 4°C with primary antibodies targeting specific proteins, including COL I (1:500), TIMP1 (1:500), ECM2 (1:20), MMP-1 (1:20), TGF-β1 (1:100), phosphorylated Smad2 (1:400), phosphorylated Smad3 (1:100), Smad2 (1:50), and Smad3 (1:50). After rinsing, the cells were incubated with secondary fluorescent antibodies for 1 h at room temperature in the absence of light. Washing with PBS followed, and then, the cells were stained with DAPI for 30 min at room temperature under light-protected conditions. Finally, the cells were washed with PBS and observed using a fluorescence microscope (BX53, Olympus).

The detection process of miR-5195-3p using a dual luciferase reporter gene assay

Using the ENCORI bioinformatics database (http://rna.sysu.edu.cn/encori/index.php), we predicted the binding sites between miR-5195-3p and mRNA sequences. Based on these predictions, we constructed plasmids containing the binding sites, precisely pGL3-LOX 3’UTR wild type (LOX 3’UTR-WT) and mutant type (LOX 3’UTR-MUT).

After 24 h of incubation, the cells were lysed, and the cell supernatant was vigorously shaken on a level shaker at room temperature for 15 min. The supernatant was collected, and we added a luciferase assay reagent using a luminometer to measure the firefly luciferase activity. We treated the same sample with a stop solution to measure marine copepod luciferase activity and used the luminometer.

The relative luciferase activity was calculated by dividing the firefly luciferase activity by the marine copepod luciferase activity.

Statistical analysis

We used SPSS 23.0 software for data processing and statistical analysis. All results are presented as mean ± standard deviation (mean ± S.D.). We used an independent-samples t-test to compare the two groups. For the comparison among multiple groups, a one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s post hoc test for comparisons. Additionally, we performed Pearson correlation analysis to examine the association between two variables. Statistical significance was considered when the P-value was less than the 0.05 threshold.

Results

Regulation of extracellular matrix metabolism in the vaginal wall of pelvic organ prolapse patients by miR-5195-3p

To investigate the potential role of miR-5195-3p in POP, we initially examined its levels in vaginal wall tissues obtained from non-POP patients and POP patients. Our qRT-PCR (Fig. 1A) and fluorescence in situ hybridization (FISH) (Fig. 1B) analyses revealed a significant upregulation of miR-5195-3p in the vaginal wall tissues of POP patients, as compared to non-POP patients. Furthermore, we assessed the expression of proteins involved in ECM metabolism in these tissues using immunohistochemistry (Fig. 1C) and immunoblotting (Fig. 1D). Our findings showed a downregulation of COL1, TIMP1, and ECM2 expression, accompanied by elevated levels of MMP-1 expression, in the vaginal wall tissues of POP patients. Subsequently, Pearson correlation analysis (Fig. 1E–H) demonstrated a negative correlation between miR-5195-3p expression and the expression of COL1, TIMP1, and ECM2 proteins in the vaginal wall tissues of POP patients, while a positive correlation was observed with MMP-1 protein expression. These results collectively suggest a potential association between the dysregulation of miR-5195-3p and an imbalance in ECM metabolism in the vaginal wall tissues of POP patients.

Figure 1

Expression of miR-5195-3p and ECM-related proteins in vaginal wall tissues of patients with pelvic organ prolapse. Note: (A) Real-time reverse transcription PCR (qRT-PCR) detection of miR-5195-3p expression in vaginal wall tissues from both non-POP and POP patients. (B) FISH examination of miR-5195-3p expression in vaginal wall tissues from both non-POP and POP patients; (C) immunohistochemistry assessment of ECM metabolism–related protein expression in vaginal wall tissues of POP patients; (D) Western blot analysis of ECM metabolism–related protein expression in vaginal wall tissues of POP patients; (E) Pearson correlation analysis between miR-5195-3p and COL-1 mRNA expression in vaginal wall tissues; (F) Pearson correlation analysis between miR-5195-3p and MMP1 mRNA expression in vaginal wall tissues; (G) Pearson correlation analysis between miR-5195-3p and TIMP1 mRNA expression in vaginal wall tissues; and (H) Pearson correlation analysis between miR-5195-3p and ECM2 mRNA expression in vaginal wall tissues. (^* indicates statistical significance compared to the control group, P < 0.05; ^# indicates statistical significance compared to the POP I-II group, P < 0.05)

To further support this hypothesis, we cultured vaginal wall fibroblasts derived from non-POP and POP patients and characterized these cells using immunofluorescent staining of vimentin, keratin, and α-SMA (Fig. 2A). Then, we transfected fibroblasts with a miR-5195-3p inhibitor and examined the expression of ECM metabolism-related proteins in these cells. Our immunoblotting and immunofluorescence results (Fig. 2B and C) demonstrated that the inhibition of miR-5195-3p increased the expression of COL-1, TIMP1, and ECM2, decreasing MMP-1 expression in the fibroblasts. In conclusion, our findings suggest that miR-5195-3p plays a regulatory role in ECM metabolism within the vaginal wall tissues of POP patients.

Figure 2

MiR-5195-3p regulates ECM metabolism in fibroblasts of vaginal wall tissues in patients with pelvic organ prolapse. Note: (A) Immunofluorescence analysis of vimentin, keratin, and α-SMA expression in fibroblasts; (B) immunoblotting assessment of ECM metabolism–related protein expression in cells; (C) immunofluorescence evaluation of ECM metabolism–related protein expression in cells. (^* indicates a significant difference compared to the control group, P < 0.05; ^# indicates a significant difference compared to the POP + inhibitor NC group, P < 0.05.)

Bioinformatics reveals key genes and mechanisms of pelvic organ prolapse

We conducted a comprehensive bioinformatics analysis to identify candidate genes and chemical components influencing POP (Fig. 3A). Based on the GSE12852 dataset, we set the screening thresholds at |log2(Fold Change)| > 1 and P < 0.05, resulting in the identification of 260 and 244 differentially expressed targets, including miR-5195-3p (Fig. 3B and C). Further screening using the GeneCards and CTD databases yielded 1921 and 3616 genes, respectively, associated with POP. By performing a Venn analysis, we identified eight essential target genes that displayed the most significant association with POP (Fig. 3D).

Figure 3

Bioinformatics identification and functional mechanisms of critical genes related to POP. Note: (A) Bioinformatics screening process for retrieving the GSE12852 dataset from GEO and comparing genes with GeneCards and CTD databases, followed by Venn analysis to identify critical genes related to pelvic POP disease; (B) volcano plot (round) of differentially expressed genes from the GSE12852 dataset using thresholds |log2(Fold Change)| > 1 and P < 0.05; (C) volcano plot (uterosacral) under the same criteria showing the distribution of differentially expressed genes in another condition; (D) Venn diagram identifying critical genes from GSE12852, CTD, and GeneCards, highlighting eight core genes associated with POP; (E) heatmap of GO functional analysis showing enrichment in biological processes (BPs), cellular components (CCs), and molecular functions (MFs) of essential genes; and (F) PPI network diagram from STRING, emphasizing LOX and COL-3A1’s central roles in POP and interactions with other genes.

Functional analysis of these eight candidate genes was performed using geneGO, and the results were ranked by p.adjust <0.01. This analysis revealed multiple BPs associated with POP, including platelet-derived growth factor binding, growth factor binding, and beta-catenin binding. Furthermore, entries related to CC and MF associated with POP were also identified. Notable CC entries included collagen-containing ECM, banded collagen fibril, and collagen trimer, while notable MF entries included a response to xenobiotic stimulus, muscle tissue development, and elastic fiber assembly (Fig. S1A–C).

To validate the enrichment of COL3A1 and LOX in multiple pathways, heatmap analysis was performed, confirming their involvement (Fig. 3E). Additionally, by analyzing the PPI relationships among the eight essential genes using the STRING database, we constructed a PPI network, highlighting the strong correlation between LOX and COL3A1 (Fig. 3F).

In conclusion, this study utilized bioinformatics methods to effectively identify critical genes closely associated with the pathological processes underlying POP. Specifically, the potential pivotal roles of COL3A1 and LOX in the pathogenesis of POP were underscored.

MiR-5195-3p regulates extracellular matrix metabolism in patients with pelvic organ prolapse by targeting LOX messenger RNA expression

LOX is a crucial ECM component that maintains its structure and function. A bioinformatics prediction on a website has suggested that miR-5195-3p may possess a binding site in the 3′ untranslated region of LOX mRNA (Fig. 4A), and this prediction was further confirmed through a dual luciferase reporter gene experiment (Fig. 4B). Additionally, the results of our study demonstrate that the expression of LOX is significantly downregulated in the vaginal wall tissue of patients with POP (Fig. 4C and D). Notably, Pearson correlation analysis reveals a negative correlation between miR-5195-3p and LOX mRNA in the vaginal wall tissue of POP patients (Fig. 4E). These findings suggest that miR-5195-3p may regulate ECM metabolism in POP patients by targeting the expression of LOX. Moreover, transfection of a miR-5195-3p inhibitor (Fig. 4F) leads to an increase in the expression of both LOX mRNA (Fig. 4G) and protein (Fig. 4H) in fibroblasts derived from the vaginal wall tissue of POP patients. Taken together, our study provides evidence that miR-5195-3p can directly target and inhibit LOX expression in the vaginal wall tissue of patients with POP.

Figure 4

The regulatory effect of miR-5195-3p on LOX mRNA expression and its expression pattern in patients with pelvic organ prolapse. Note: (A) Bioinformatics analysis predicted a binding site between miR-5195-3p and the 3′ untranslated region of LOX mRNA, as shown in the schematic diagram. (B) The interaction between miR-5195-3p and LOX mRNA was functionally validated using the dual luciferase reporter gene assay. Compared to the miR-NC (negative control) group, the experimental group showed significant differences at P < 0.05, indicating a significant binding. (C) Real-time reverse transcription PCR (qRT-PCR) analysis revealed differential expression of LOX mRNA in vaginal wall tissues of patients without pelvic organ prolapse (non-POP) and pelvic POP patients. (D) Western blot analysis showed differential expression of LOX protein in vaginal wall tissues of non-POP patients and POP patients. Significant differences were observed compared to the normal group at P < 0.05 and to the POP I-II stage group at ^#P < 0.05. (E) Pearson correlation analysis demonstrated a negative correlation between miR-5195-3p and LOX mRNA expression levels in vaginal wall tissues of POP patients. (F) The expression level of miR-5195-3p in fibroblasts was analyzed using qRT-PCR. (G) QRT-PCR analysis assessed the impact on LOX mRNA expression in fibroblasts after transfection with a miR-5195-3p inhibitor. All cell experiments were repeated three times. (H) Western blot analysis evaluated the transfection effect of the miR-5195-3p inhibitor on LOX protein expression levels in fibroblasts. ^* represents a comparison with the control group at P < 0.05; ^# represents a comparison with the POP + inhibitor NC group at P < 0.05.

MiR-5195-3p regulates extracellular matrix metabolism in vaginal wall tissues of pelvic organ prolapse patients through targeting and inhibiting LOX

We investigated the regulatory role of miR-5195-3p in the metabolism of ECM proteins in the vaginal wall tissue of patients with POP by targeting LOX. Using qRT-PCR, we found that the miR-5195-3p inhibitor effectively suppressed the RNA expression levels of miR-5195-3p (Fig. 5A) while significantly restoring LOX RNA levels (Fig. 5B). Our Western blot analysis further confirmed that the use of the miR-5195-3p inhibitor significantly increased LOX protein levels (Fig. 5C). Additionally, transfection with the miR-5195-3p inhibitor upregulated the expression of COL-1, TIMP1, and ECM2 proteins while reducing the expression of MMP-1 protein in fibroblasts derived from the vaginal wall of POP patients. Immunofluorescence results further validated the upregulation of COL-1, TIMP1, and ECM2 proteins, along with the reduction of MMP-1 protein caused by the miR-5195-3p inhibitor (Fig. 5D). Moreover, inhibition of LOX reversed the effects of the miR-5195-3p inhibitor (Fig. 5C and D). Additionally, we replicated these experiments using the 3 T3-L1 mouse fibroblast cell line, further confirming the role of miR-5195-3p in ECM remodeling and demonstrating that LOX can reverse the effects of the miR-5195-3p inhibitor (Fig. S2A-D).

Figure 5

MiR-5195-3p regulates ECM metabolism in the vaginal wall tissue of POP patients by targeting and inhibiting LOX. Note: (A) MiR-5195-3p expression in fibroblasts was assessed using qRT-PCR. (B) QRT-PCR quantified LOX mRNA expression in fibroblasts. (C) The LOX and ECM metabolism–related protein levels in fibroblasts were determined via Western blot analysis. (D) Immunofluorescence was employed to investigate the expression of ECM metabolism–related proteins in fibroblasts. Statistical analysis revealed significant differences: ^* denotes a significant difference compared to the POP + inhibitor NC group (P < 0.05), while ^# indicates a significant difference compared to the POP + miR inhibitor + si-NC group (P < 0.05).

These compelling findings suggest that miR-5195-3p plays a crucial role in regulating ECM metabolism in the vaginal wall tissue of POP patients through specific targeting and inhibition of LOX.

LOX regulates ECM metabolism of the vaginal wall tissues in pelvic organ prolapse patients through the TGF-β1 Signaling pathway

TGF-β1 plays a crucial role in promoting the synthesis of ECM. However, inhibiting the activity of LOX weakens the TGF-β1 signaling pathway. In this study, we aimed to investigate whether the TGF-β1 signaling pathway is involved in regulating ECM metabolism in vaginal wall tissues of patients with pelvic POP.

To begin, we assessed the protein expression of the TGF-β1 signaling pathway in vaginal wall tissues using Western blot analysis (Fig. 6A). The results revealed that TGF-β1, p-Smad2, and p-Smad3 were significantly downregulated in the vaginal wall tissues of POP patients. This finding indicates that the TGF-β1 signaling pathway is dysregulated in the vaginal wall tissues of individuals with POP.

Figure 6

LOX regulates ECM metabolism in POP patient vaginal wall tissues through the TGF-β1 signaling pathway. Note: (A) The protein expression levels of TGF-β1 signaling pathway–related proteins were assessed in the vaginal wall tissue of patients with pelvic POP using Western blot analysis. Significantly higher levels of these proteins were observed compared to the standard control group (P < 0.05). Furthermore, a statistically significant difference was found between the POP I-II and regular control groups (^#P < 0.05). (B) Western blot analysis investigated the expression of proteins involved in ECM metabolism in fibroblasts. (C) Immunofluorescence assays were conducted to evaluate the expression of proteins related to ECM metabolism in fibroblasts. A significant difference was observed when comparing the POP + vector group to the standard control group (P < 0.05). A statistically significant difference was observed between the POP + ov-LOX + Dimethyl Sulfoxide (DMSO) group and the POP + vector group (^#P < 0.05).

To further explore the impact of LOX on the expression of ECM-related proteins in vaginal wall fibroblasts of POP patients and its regulation through the TGF-β1 signaling pathway, we performed Western blot analysis (Fig. 6B) and immunofluorescence staining (Fig. 6C). The results demonstrated that overexpression of LOX increased the expression of COL-1, TIMP1, and ECM2 in vaginal wall fibroblasts of POP patients while decreasing the expression of MMP-1. Furthermore, treatment with Disitertide, a TGF-β1 inhibitor, resulted in reduced expression of COL-1, TIMP1, and ECM2 and increased expression of MMP-1 in vaginal wall fibroblasts of POP patients.

These findings indicate that the LOX/TGF-β1 signaling pathway regulates the ECM metabolism of vaginal wall fibroblasts in patients with POP.

MiR-5195-3p regulates ECM metabolism through the LOX/TGF-β1 Axis

To investigate the potential role of miR-5195-3p in regulating the TGF-β1 signaling pathway by targeting LOX inhibition, we conducted a study to examine the effects of silencing LOX and transfecting a miR-5195-3p inhibitor on the levels of TGF-β1 signaling pathway-related proteins in vaginal wall fibroblasts obtained from patients with pelvic POP. The results of our Western blot analysis (Fig. 7A) revealed that inhibition of miR-5195-3p increased the expression levels of TGF-β1, p-Smad2, and p-Smad3 in vaginal wall fibroblasts from patients with POP. Interestingly, when we silenced LOX, the stimulatory effect of the miR-5195-3p inhibitor on the TGF-β1 signaling pathway was reversed in these fibroblasts. These findings suggest that miR-5195-3p negatively regulates the TGF-β1 signaling pathway by inhibiting LOX expression.

Figure 7

MiR-5195-3p regulates ECM metabolism in the vaginal wall tissue of POP patients through the LOX/TGF-β1 signaling pathway. Note: (A) The expression of proteins related to the TGF-β1 signaling pathway in fibroblasts was analyzed using Western blot. Statistical analysis revealed a significant difference (P < 0.05) compared to the POP + inhibitor NC group. A significant difference (^#P < 0.05) was observed compared to the POP + miR-inhibitor + si-NC group. (B) The expression of proteins related to ECM metabolism in fibroblasts was analyzed using Western blot. (C) Immunofluorescence analysis was conducted to examine the expression of proteins related to ECM metabolism in fibroblasts. Statistical analysis indicated a significant difference (P < 0.05) compared to the POP + inhibitor NC group. Furthermore, a significant difference (^#P < 0.05) was observed compared to the POP + miR-inhibitor + si-NC group. A significant difference (&P < 0.05) was also observed compared to the POP + miR-inhibitor + si-LOX + vector group.

Our study also explored the effects of miR-5195-3p/LOX/TGF-β1 axis on ECM metabolism in vaginal wall fibroblasts from patients with POP. Western blot analysis (Fig. 7B) and immunofluorescence staining (Fig. 7C) were performed to examine the relationship between the blockade of the miR-5195-3p/LOX/TGF-β1 signaling pathway and ECM metabolism imbalance in these fibroblasts. The results demonstrated that transfection of the miR-5195-3p inhibitor increased the expression of COL-1, TIMP1, and ECM2 in vaginal wall fibroblasts from patients with POP, while it decreased the expression of MMP-1. Conversely, LOX silencing decreased the expression of COL-1, TIMP1, and ECM2 but increased the expression of MMP-1. Notably, the overexpression of TGF-β1 reversed the effects of LOX silencing on the expression of ECM metabolism–related proteins.

To further confirm the crucial role of miR-5195-3p in POP, we established a postpartum injury-induced POP rat model (Fig. S3A). By administering the miR-5195-3p inhibitor in vivo, we reduced miR-5195-3p expression levels in the model. Previous studies have reported that MMP2 and MMP9 are involved in the pathophysiology of pelvic floor dysfunction [65]; therefore, we used MMP2/9 as indicators of improvement in the POP model. Immunohistochemistry results indicated that, following the inhibition of miR-5195-3p, the levels of MMP2/9 in the tissue decreased (Fig. S3B), promoting ECM restoration and improving POP prognosis. Additionally, to verify that miR-5195-3p exerts its regulatory function through the LOX/TGF-β1 axis, we also assessed LOX and TGF-β levels via immunohistochemistry (Fig. S3B). The results showed that after miR-5195-3p inhibition, LOX levels increased, while TGF-β levels decreased. These findings collectively demonstrate that the miR-5195-3p/LOX/TGF-β1 axis can regulate ECM metabolism in vaginal wall tissue fibroblasts from patients with POP.

Discussion

Pelvic POP is a prevalent condition affecting women, characterized by the protrusion of pelvic organs through the pelvic floor and vaginal wall [1, 66]. It commonly affects older women, with approximately 6% of symptomatic patients aged 20–29 and 31% aged 50–59. Moreover, about 50% of patients affected by POP are 80 years old or older [67, 68]. As life expectancy increases and the population ages, POP is expected to become a significant health concern [69]. Projections indicate that by 2050, the prevalence of symptomatic POP in the United States will rise to 46% [70]. Understanding the risk factors associated with POP is crucial as they contribute to the weakening of pelvic floor connective tissue and collagen, ultimately leading to organ prolapse through the vaginal wall and pelvic floor [71–73]. Currently, surgery is the primary treatment option for POP, aiming to restore the anatomical structure of the vagina and surrounding organs, thereby alleviating pelvic floor–related symptoms [72]. However, surgical recurrence rates remain high, and complications are frequent [74–78]. Therefore, an in-depth investigation of the molecular mechanisms underlying POP pathogenesis is essential for developing effective treatment methods.

MiRNAs, consisting of short, single-stranded RNA molecules typically about 22 nucleotides long [79], have been found to play a crucial role in various diseases, making them potential therapeutic targets [80, 81]. The main approaches for utilizing miRNAs as therapeutic agents involve restoring underexpressed miRNAs and inhibiting overexpressed miRNAs [82, 83]. There are few studies on the treatment of POP with miRNAs; however, there is a growing body of research on targeting miRNAs in cervical and uterine-related diseases. For example, Jeon et al. provided evidence that miR-30d and miR-181a are highly expressed in the uterosacral ligament of POP patients and target HOXA11 expression, thereby inhibiting collagen synthesis [84–86]. In another study, Shi et al. found elevated levels of miR-221 and miR-222 in the cervical part of the uterosacral ligament in POP patients, which target ERα expression [87, 88]. He et al. demonstrated that miR-92 is significantly upregulated in the uterosacral ligament tissue of POP patients compared to non-POP patients and targets ERβ1 expression [89]. Furthermore, Zhao et al. revealed that miR-138 is overexpressed in bone marrow mesenchymal stem cells of patients with pelvic floor dysfunction and POP, leading to targeted inhibition of FBLN5 and resulting in inhibited cell proliferation, increased IL-1β expression, and decreased elastic protein expression [90]. These studies suggest that miRNAs not only play a role in POP but also influence cervical and uterine function by regulating target genes in the uterosacral ligament, indicating their multiple regulatory roles in these organs. In summary, miRNAs hold promise as potential therapeutic targets for treating POP. There have been no reports on the role of miR-5195-3p in the regulation of POP. Although progress has been made in the study of other miRNAs, there are no reports on the regulatory role of miR-5195-3p in POP, leaving space for further exploration of its potential mechanisms in POP.

The support of the pelvic organs is primarily provided by the pelvic floor muscles, pelvic fascia, and ligaments such as the primary and uterosacral ligaments [17, 91, 92]. These structures are composed mainly of connective tissue containing fibroblasts, which produce various molecules that comprise the ECM, including structural proteins, matrix adhesive molecules, and proteoglycans. Collagen and elastin are critical components for maintaining the integrity of the pelvic floor [93, 94]. The mechanical strength of collagen relies on cross-linking, involving enzymatic-driven lysine oxidation and non-enzymatic glycation processes [95]. LOX is the enzyme initiating the enzymatic cross-linking process. LOX oxidizes lysine or hydroxylysine residues, creating irreversible bonds with natural lysine or hydroxyproline residues on other collagen molecules. This cross-linking enhances the physical strength and resistance to degradation by collagenases [96–99].

LOX belongs to the lysyl oxidase family, a copper-dependent amine oxidase with highly conserved catalytic domains, including the lysyl tyrosylquinone cofactor (LTQ) and copper-binding sites [100, 101]. Its principal function is to catalyze the oxidative deamination of primary amino groups to form active aldehyde groups [102–104].

Previous studies have reported that LOX is involved in mediating other pelvic floor dysfunction diseases. Compared to normal individuals, patients with POP exhibit increased methylation levels in the LOX gene promoter region, which inhibits LOX gene expression [105]. The TT homozygous polymorphism in the rs1048661 and rs2165241 regions of the LOX gene may also be associated with the occurrence of stress urinary incontinence [106]. Furthermore, LOX is closely linked to the development and progression of various other diseases. Saatci et al. demonstrated that LOX increased the levels of ITGA5 and its ligand fibronectin in triple-negative breast cancer cells, promoting collagen cross-linking and fibronectin fiber assembly. This resulted in reduced penetration of chemotherapy drugs and increased chemoresistance [107]. Nguyen et al. revealed that LOX induced nuclear translocation by increasing c-Fos expression and binding to the IL-6 promoter region. This led to upregulation of IL-6 expression, ECM synthesis, and pulmonary fibrosis [108]. Varona et al. showed that high expression of LOX in vascular smooth muscle cells induced fibronectin deposition and collagen cross-linking [109, 110]. These studies highlight the crucial role of LOX in the synthesis and stability of the extracellular matrix in various diseases.

The role of LOX and TGF-β1 has been revealed in the pathogenesis of various cervical and uterine-related diseases [98, 111]. Through biochip sequencing analysis, TGF-β can regulate LOX gene expression via pathways such as Smad, JNK, and MAPK. Specifically, TGF-β induces LOX expression, promoting the cross-linking of elastic fibers and collagen in the ECM of POP patients. Reports on TGF-β mRNA levels in POP patients suggest no significant difference compared to normal controls, while LOX levels are notably decreased [55]. However, it has been reported that TGF-β can upregulate LOX expression [112], and TGF-β delays the degradation of ECM components [58]. Additionally, TGF-β activates TIMP-2 synthesis in the uterosacral ligament and inhibits MMP2/9 activity, reducing ECM loss. The activation of the TGF-β signaling pathway involves signal transduction from the receptor to the nucleus, initiating the transcription of ECM components. For example, in uterine fibroids, TGF-β promotes the excessive production of glycosaminoglycan-rich multifunctional variants, leading to ECM dysregulation [113].

Our research indicates that inhibiting miR-5195-3p in fibroblasts of the vaginal wall tissue in POP patients leads to an imbalance in ECM metabolism, manifested by an increase in the expression of COL-1, TIMP1, and ECM2 proteins, and a decrease in MMP-1 protein expression. This effect is reversed by silencing LOX, suggesting that miR-5195-3p regulates ECM metabolism by targeting and inhibiting LOX expression. Furthermore, overexpression of TGF-β1 reverses the silencing effect of LOX, indicating that miR-5195-3p inhibits the LOX-mediated inhibition of the TGF-β1 signaling pathway. These results suggest that targeting miR-5195-3p/LOX could be a promising therapeutic option for treating POP.

From a scientific perspective, this study reveals the crucial role of miR-5195-3p in the pathogenesis of POP, particularly in its regulation of ECM metabolism, providing new knowledge for a deeper understanding of the molecular biology basis of POP. From a clinical perspective, this finding offers possibilities for developing therapeutic strategies targeting miR-5195-3p, especially in the non-surgical treatment of POP. Additionally, understanding the role of miR-5195-3p in ECM metabolism may contribute to preventing or alleviating POP symptoms and improving patients’ quality of life.

The limitations of this study mainly lie in the sample size and experimental design. Firstly, the sample size is relatively small, which may affect the generalizability and statistical significance of the results. Secondly, the study primarily focuses on the role of miR-5195-3p in the pathogenesis of POP and needs to explore other potential molecular mechanisms comprehensively. Furthermore, the study relies on in vitro experimental models, which may only partially reflect the complex environment and interactions within the human body.

Future research could focus on expanding the sample size to enhance the generalizability and reliability of the findings. Additionally, it is necessary to conduct more in vivo experiments and clinical trials to validate the role of miR-5195-3p in the pathogenesis of POP and explore potential therapeutic approaches targeting miR-5195-3p. In our study, we selected a rat POP model for the in vivo experiments. We acknowledge that, compared to large animal models such as sheep, rodent models may have relatively lower translational relevance and persuasive power. Furthermore, research should continue to investigate other molecular mechanisms of POP to fully understand the pathogenesis of this complex disease and develop more effective preventive and treatment strategies.

In summary, this study demonstrates that miR-5195-3p is significantly upregulated in the vaginal wall tissue of patients with POP and directly targets LOX by binding to the 3′ untranslated region of its mRNA, inhibiting its expression. miR-5195-3p was also negatively correlated with the expression of ECM metabolism–related proteins, suggesting its crucial role in ECM regulation. Furthermore, we showed that modulating LOX expression impacts the TGF-β1 signaling pathway, further affecting ECM metabolism (Fig. 8). These results indicate that miR-5195-3p is essential in POP pathogenesis through the LOX/TGF-β1 axis, providing a potential molecular target for future therapeutic strategies.

Figure 8

The function of miR-5195-3p in pelvic organ prolapse and its regulation on the LOX/TGF-β1 signaling axis.

Acknowledgment

Not applicable.

Conflict of interest: The authors have declared that no conflict of interest exists.

Data availability

The data that support the findings of this study are available on request from the corresponding author upon reasonable request.

Author contributions

Hao Zhang and Xinlu Wang contributed equally to the experimental design, data acquisition, and analysis. Meng Dong was responsible for conducting the molecular biology experiments, including qRT-PCR, FISH, and Western blotting. Jie Wang contributed to bioinformatics analysis and the interpretation of results. Weidong Ren supervised the study, provided critical revisions, and served as the corresponding author. All authors contributed to manuscript writing and approved the final version.

Ethical statement

This study was approved by the Ethics Committee of Shengjing Hospital, China Medical University (2022PS515K), and the animal ethics approval was obtained with the number CMUXN2022301. A total of 30 patients who underwent total hysterectomy were enrolled in the study, with written informed consent obtained from the participants or their families. All procedures involving animals were reviewed and approved by the appropriate ethics committee and were conducted in accordance with the institution’s guidelines for animal care and use.

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