| Objective: Pelvic organ prolapse(POP)is a type of disease that is caused by the weakness of pelvic floor support for various reasons,resulting in a decrease in the position of the pelvic organs and abnormal pelvic floor function,mainly manifesting as prolapse or bulging of the anterior pelvic organs(bladder,urethra,anterior vaginal wall),middle pelvic organs(uterus,vaginal roof),and posterior pelvic organs(posterior vaginal wall,rectum).The prevalence of POP increases significantly with age,and although it is a non-fatal disease,the social and psychological distress it brings affects the quality of life of patients to varying degrees.The pathogenesis of POP is not clear.At present,molecular target therapy has become a hot spot for clinical research,and the search for effective molecular therapeutic targets has significant clinical significance for the targeted treatment of POP.The supporting structures of female pelvic organs mainly include connective tissues such as pelvic floor nerves,muscles,ligaments and fascia.Fibroblasts in connective tissues secrete large amounts of extracellular matrix(ECM),which consists of structural proteins(collagen and elastin),matrix adhesion molecules(fibronectin and laminin),and proteoglycans.Studies have shown that ECM breakdown weakens the strength of the supporting structures and contributes to POP development.Thus,ECM content and dysfunction are the molecular and biochemical basis for POP development.Micro RNAs(mi RNAs)are non-coding RNAs,approximately 22 nucleotides in length.They are endogenous RNA molecules that complement and bind to regulatory sites in the 3’ untranslated region of target messenger RNA(messenger RNA,m RNA),resulting in translation inhibition or m RNA degradation.Mi RNA plays a role in the regulation of ECM metabolism.Mi R-5195-3p was discovered by deep sequencing of small RNA in human acute lymphoblastic leukemia in 2011.At present,the research on it mainly focuses on the field of tumor and plays an anti-cancer role.The regulation of mi R-5195-3p on ECM metabolism and the role of mi R-5195-3p in the process of POP have not been reported.The lysyl oxidase(LOX)family is a secreted copper-dependent amine oxidase that catalyzes the oxidative deamination of lysine and hydroxylysine residues to form highly reactive peptidyl semialdehydes,these peptidyl semialdehydes condense together to form intramolecular and intermolecular bonds.Thereby promoting the formation of intramolecular and intermolecular cross-connections of ECM components and the development of tissue-specific insoluble matrices,helping the ECM to exert its local structure and signaling environment during development,tissue maintenance and repair,and various pathological processes,Functions that regulate cell fate,adhesion,proliferation,differentiation,migration and communication.LOX and transforming growth factor β(TGF-β)have a mutual regulatory effect.Downregulation of LOX expression can lead to reduced trophoblast migration and invasion by inhibiting the TGF-β1/SMAD family member(Smad)3/collagen pathway.In a bleomycin-induced model of idiopathic pulmonary fibrosis,LOX expression was upregulated,and LOX knockdown or inhibition attenuated pulmonary fibrosis,the mechanisms of which were associated with reduced TGF-β signaling and myofibroblast accumulation.In conclusion,this study aimed to explore the role of mi R-5195-3p in the occurrence of POP,mainly to explore its effect on the ECM metabolism of vaginal wall tissue in patients with POP,and to further study the targeting relationship between mi R-5195-3p and LOX.The role of LOX/TGF-β signaling pathway in mi R-5195-3p in regulating ECM metabolism in vaginal wall tissue of patients with POP provides new ideas for the treatment and diagnosis of POP.Methods:1.To explore the effect of mi R-5195-3p on ECM metabolism in POP patients:The vaginal wall tissues of POP patients were collected,and the expression of mi R-5195-3p in the vaginal wall tissues was detected by real-time quantitative PCR(q RT-PCR)and fluorescence in situ hybridization(FISH).The expression of ECM metabolism-related proteins in the vaginal wall tissues was detected by immunohistochemistry and Western blot,and the vaginal wall fibroblasts of POP patients were isolated and identified by immunofluorescence.Vaginal wall fibroblasts were transfected with mi R-5195-3p inhibitor,and the expression of mi R-5195-3p in cells was detected by q RT-PCR,cell proliferation was detected by cell counting kit-8(CCK-8),cell apoptosis was detected by flow cytometry and the expression of ECM metabolism-related proteins in cells was detected by Western blot and immunofluorescence.2.To explore whether mi R-5195-3p affects ECM metabolism in POP patients by targeting LOX: Vaginal wall tissues of POP patients were collected,and LOX m RNA expression in vaginal wall tissues was detected by q RT-PCR.LOX protein expression in vaginal wall tissues of POP patients was detected by Western blot and immunohistochemistry.Vaginal wall fibroblasts of POP patients were isolated and transfected with LOX overexpressed plasmid.The expression of LOX m RNA in cells was detected by q RT-PCR,the expression of LOX protein and ECM metabolism-related proteins in vaginal wall tissues was detected by Western blot and immunofluorescence,cell proliferation was detected by CCK-8,cell apoptosis was detected by flow cytometry and the potential binding sites between mi R-5195-3p and LOX m RNA in the 3’untranslated region were predicted by bioinformatics website.The vaginal wall fibroblasts were transfected with mi R-5195-3p inhibitor,and the LOX m RNA expression was detected by q RT-PCR,and the LOX protein expression was detected by Western blot.Cell proliferation was detected by CCK-8,cell apoptosis was detected by flow cytometry.The expression of ECM metabolism-related proteins in vaginal wall tissues was detected by Western blot and immunofluorescence.3.To investigate whether mi R-5195-3p affects ECM metabolism in POP patients by regulating TGF-β1 expression through targeting LOX.The vaginal wall tissues of POP patients were collected,and TGF-β1 protein expression was detected by Western blot.The vaginal wall fibroblasts of POP patients were isolated and transfected into the vaginal wall cells with mi R-5195-3p inhibitor or LOX overexpression plasmid.Cell proliferation was detected by CCK-8,cell apoptosis was detected by flow cytometry.The expression of TGF-β1 signaling pathway related proteins in cells was detected by Western blot,and the expression of metabolism-related proteins in ECM cells after TGF-β1 overexpression was detected by Western blot and immunofluorescence.Results: 1.The expression of mi R-5195-3p and matrix metalloproteinase-1(MMP1)was high in the vaginal wall tissues of patients with POP,while the expression of collagen I(COL1),tissue inhibitor of metalloproteinase 1(TIMP1)and extracellular matrix protein2(ECM2)was low.The transfection of mi R-5195-3p inhibitor decreased cell apoptosis rate and the expression of mi R-5195-3p and MMP1 in the vaginal wall fibroblasts of patients with POP,and increased the cell proliferation activity and expression of COL1,TIMP1 and ECM2.2.LOX expression was low in vaginal wall tissue of POP patients.Transfection of LOX overexpression plasmid increased the cell proliferation activity and expression of LOX,COL1,TIMP1 and ECM2 in vaginal wall fibroblasts of POP patients,decreased cell apoptosis rate and the expression of MMP-1 protein,mi R-5195-3p targeted binding to the 3’ untranslated region of LOX m RNA,and inhibited the expression of LOX.Transfection of LOX si RNA reversed the effect of mi R-5195-3p inhibitor on cell proliferation,apoptosis and the expression of ECM metabolism-related proteins in vaginal wall fibroblasts of POP patients.3.TGF-β1,p-Smad2,and p-Smad3 proteins were low expressed in vaginal wall tissue of patients with POP,and transfection with mi R-5195-3p inhibitor or overexpression of LOX increased the protein expressions of TGF-β1,p-Smad2,and p-Smad3 in vaginal wall fibroblasts of patients with POP.The transfection of LOX si RNA reversed the effect of mi R-5195-3p inhibitor and overexpression of LOX on the expression of TGF-β1 signaling pathway related proteins in vaginal wall fibroblasts of POP patients.The transfection of TGF-β1 overexpression plasmid increased the cell proliferation activity and expression of COL1,TIMP1 and ECM2,and decreased cell apoptosis rate and the expression of MMP-1.Overexpression of TGF-β1 reversed the effect of LOX si RNA transfection on cell proliferation,apoptosis and the expression of ECM metabolism-related proteins in vaginal wall fibroblasts from patients with POP.Conclusion: Mi R-5195-3p regulates the imbalance of ECM metabolism in vaginal wall tissues of POP patients by targeting the expression of LOX and inhibiting the TGF-βsignaling pathway. |
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