Expression And Correlation Of DKK1 And Wnt/β-catenin Signaling Pathway In The Utero-sacral Ligament Of Female Pelvic Organ Prolapse Patients

Objective:In this experiment,we researched the expression and correlation of DKK1,Wnt16,β-catenin,collagen Ⅰ（COL Ⅰ）and collagen Ⅲ（COL Ⅲ）in the utero-sacral ligament tissues of POP and non-POP patients,in which Wnt16 andβ-catenin are key proteins in the Wnt/β-catenin signaling pathway.This study was to explore the potential role of DKK1 and Wnt/β-catenin signaling pathway in the development of pelvic organ prolapse,and to provide a new theoretical basis for the diagnosis and treatment of the POP.Methods:1.Thirty patients with pelvic organ prolapse admitted to the Department of Obstetrics and Gynecology of the Second Hospital of Shanxi Medical University from October 2021 to October 2022 were selected as the POP group.According to pelvic organ prolapse quantitation（POP-Q）,all patients in POP group had graded Ⅲ-Ⅳ prolapse.Another thirty patients who were hospitalized for non-POP benign diseases in the gynecology department of the hospital during the same period were selected as the control group.All patients underwent laparoscopic or total transabdominal hysterectomy.After obtaining the consent of the patients,the utero-sacral ligament tissues were taken during the operation and preserved for subsequent studies.2.High-throughput sequencing of the utero-sacral ligament tissues from four patients in the POP group and five patients in the control group to screen for differential genes between POP and non-POP patients.3.Hematoxylin-eosin staining was used to confirm and observe the histological characteristics of utero-sacral ligament,and Masson staining was used to observe the number,morphology and arrangement of collagen fibers in the utero-sacral ligament.4.The messenger RNA expression of DKK1,Wnt16 andβ-catenin was detected by polymerase chain reaction,in which Wnt16 andβ-catenin were key proteins of the Wnt/β-catenin signaling pathway.5.The protein expression of Wnt16,β-catenin,DKK1,COL Ⅰ and COL Ⅲ was detected by Western-blot,in which Wnt16 andβ-catenin were key proteins of the Wnt/β-catenin signaling pathway.In the POP group,the correlation of DKK1 with Wnt16 andβ-catenin was analyzed.6.SPSS24.0 and Graph Pad Prism 6 software were used to analyze the data.Measurement data were described as mean±standard deviation（（?）±S）.Independent sample t test was used to compare the measurement data between the two groups.The correlation between DKK1 and Wnt16 was analyzed by Pearson correlation coefficient,and the correlation between DKK1 and β-catenin was similarly.Results:1.There were 222 significantly differentially expressed genes in the sequencing results,of which 124 genes were significantly up-regulated in the POP group and 98 genes were significantly down-regulated in the POP group,and the differentially expressed genes involved various proteins and metabolic pathways.DKK1 was significantly upregulated in POP patients（log₂Fold Change:3.5188 and padj<0.05）and Wnt16 was significantly downregulated in POP patients （log₂Fold Change:-1.8111 and padj<0.05）.2.Hematoxylin-eosin staining confirmed that the tissue was ligamentous tissue.Under the microscope,the number of fibroblasts was decreased in the utero-sacral ligament tissue of POP group.Collagen fibers were small and loosely connected,and collagen fibers lost their normal alignment with smooth muscle bundles.In contrast,there were more fibroblasts in the control group,and the collagen fibers were thick and continuous with more regular arrangement.3.PCR experiments revealed that DKK1 expression in the utero-sacral ligament tissues of patients in the POP group was higher than that in the control group,while mRNA expression of Wnt16 andβ-catenin in the POP group was lower than that in the control group.4.Western blot experiments confirmed that DKK1 expression in the utero-sacral ligament of POP patients（0.85±0.07）was significantly higher than that of controls（0.34±0.02）.While the relative protein expressions of Wnt16,β-catenin,COL Ⅰ and COL Ⅲ in the POP group（0.19±0.03,1.01±0.41,0.38±0.06,0.71±0.09）were respectively lower than those in the control group（1.09±0.19,2.92±0.08,1.05±0.37,1.43±0.13）.All date were statistically significant（p<0.05）.5.DKK1 had a negative correlation with Wnt16 in the Wnt/β-catenin signaling pathway in the POP group of patients（r=-0.402,p=0.021）.Similarly there was a negative correlation between DKK1 andβ-catenin（r=-0.685,p=0.015）.Conclusion:1.DKK1 and Wnt16 were both significantly differentially expressed genes between POP patients and non-POP patients.The expression of DKK1 increased,but the expression of Wnt16 andβ-catenin was decreased in the utero-sacral ligament tissue of POP patients.2.Imbalance of collagen metabolism is inseparable from the occurrence of POP.3.The disorder of collagen metabolism in POP patients may be related to the inhibition of Wnt/β-catenin signaling pathway.4.DKK1 may inhibit Wnt/β-catenin signaling pathway,which may be one of the causes of pelvic floor dysfunction or one of the reasons for the aggravation of pelvic floor dysfunction.