| Objective: Pelvic organ prolapse(POP)is the abnormal position and function of organs caused by the descending of pelvic organs caused by abnormal pelvic floor muscles and fascia.Defecation and sexual dysfunction affect the quality of life of patients to varying degrees.In recent years,the researches on the risk factors of pelvic organ prolapse are of great significance to the discovery of its etiology,but its pathogenesis has not been clearly elucidated.Because POP seriously affects the psychological and social activities of adult women,and it can bring personal effects,great social and economic pressure.Therefore,research on its pathogenesis is helpful for the prevention,diagnosis and treatment of pelvic organ prolapse.The purpose of this study was to explore the expressions of mi R-23a-3p,EGR1 and NFκB signaling pathways in the vaginal wall tissues of POP patients,analyze their relationship with collagen metabolism,and speculate their regulatory role and mechanism in the occurrence and development of POP,to provide a theoretical basis for the prevention and treatment of POP.POP is a disease in which the repair and reconstruction of pelvic floor tissue fails to meet the requirements after long-term stress injury,resulting in dysfunction.We call the situation where the tissue is in a state of stress or dysfunction as atypical inflammation,so it can be considered that POP also has atypical inflammation.The nuclear factor κB(NFκB)signaling pathway is an important inflammatory-related pathway,and can also sense stress stimuli.At the same time,studies have shown that it plays an important role in the formation of collagen fibers,and also affects the content of factors related to collagen metabolism,such as matrix metalloproteinase(MMPs)and tissue inhibitor of metalloproteinase(TIMPs).Transcriptional activity is the main function of early growth response factor 1(EGR1),which widely affects apoptosis,remodeling,inflammation,and ECM formation.In recent years,non-coding RNAs have emerged as a research hotspot for many diseases,among which micro RNAs(mi RNAs)are currently the most studied non-coding RNAs.Previous studies have shown that there were multiple mi RNAs related to POP.Through Targetscan,Starbase,mi RDB and other websites,it was predicted that mi R-23a-3p is an upstream regulatory molecule of EGR1.Studies have shown that mi R-23a-3p is associated with tumors,fibrosis,and cell proliferation and apoptosis related to death,and EGR1 is predicted to bind the promoter region of NFκB1.Therefore,we plan to use molecular biology research methods on the basis of previous research to detect the differences in the expression levels of mi R-23a-3p,EGR1,MMP9,TIMP1,collagen type I and collagen type III between the POP group and the control group.And according to the specific regulatory mechanism in cells,the results will provide a theoretical basis for the mechanism,diagnosis and treatment of POP.Methods: 1.The vaginal wall tissue in the discarded tissue of patients undergoing total hysterectomy in Shengjing Hospital Affiliated to China Medical University from January2016 to March 2021 was collected.There were 30 cases in the control group and 30 cases in the POP group.All are grades III-IV.2.After the collected tissue samples were processed,Western blot,immunohistochemistry,Masson staining and RT-q PCR were used to detect mi R-23a-3p,EGR1,MMP9,TIMP1,collagen type I and collagen type III in vaginal wall tissue of the two groups of patients.We would detect whether there are differences in the expression levels of type I collagen,type III collagen and NFκB signaling pathway-related molecules.3.3 cases of POP tissue and 3 cases of control tissue were taken from vaginal wall tissue,and fibroblasts were grown by adherence method and differential time adherence method.The primary culture and purification of fibroblasts were identified by immunocytochemical method,and the proliferation status of two groups of fibroblasts was detected by CCK8 method.4.WB method was used to detect the expression of EGR1,NFκB signaling pathway-related molecules and collagen metabolism-related proteins in the two groups of cells.5.Using cell transfection technology,with EGR1 knocked down and overexpressed,the expression of collagen metabolism-related proteins was detected by WB method,and the changes of cell proliferation and apoptosis were detected by CCK8 method and flow cytometry.After knockdown and overexpression of EGR1,the changes of key proteins in the NFκB signaling pathway,as well as cell proliferation and apoptosis were observed.The interaction between EGR1 and NFκB1 protein was detected by co-immunoprecipitation method.6.We used cell transfection technology to knock down and overexpress mi R-23a-3p,detected the expression of EGR1 by WB method,and detected the changes of cell proliferation and apoptosis by CCK8 method and flow cytometry.7.And we also used the dual-luciferase reporter gene method to detect whether there was a direct effect.8.SPSS23.0 software was used to analyze the experimental data,and t test and one-way anova were used to analyze the results.P<0.05 was considered statistically significant.Results: 1.Masson staining showed that the collagen fibers in the POP group were broken,disordered and loose,while the collagen fibers in the control group were arranged tightly and orderly.Compared with the control group,the expression of EGR1 in the vaginal wall tissue of the patients in the POP group increased,and the expression of mi R-23a-3p decreased.The expression of MMP9 was higher in the POP group,while the expression of TIMP1 was lower.The expression levels of type Ⅰ collagen and typeⅢ collagen in vaginal wall tissue of patients in the POP group were lower.2.The proliferation of vaginal wall fibroblasts in the POP group was significantly lower than that in the control group,the proportion of apoptotic cells was higher than that in the control group,and the proportion of senescent cells was higher.The expression levels of type Ⅰ collagen,type Ⅲ collagen and TIMP1 in fibroblasts in POP group were lower than those in the control group,while the expression levels of EGR1 and MMP9 were higher than those in the control group.3.After overexpression of EGR1,the proliferation rate of fibroblasts decreased,the proportion of apoptosis increased,the expression of MMP9 increased,and the expressions of TIMP1,type Ⅰ collagen and type Ⅲ collagen decreased,while after knockdown of EGR1,the proliferation of fibroblasts accelerated,and the proportion of apoptosis decreased,the expression of MMP9 decreased,and the expressions of TIMP1,type Ⅰ collagen and type Ⅲ collagen increased.The expressions of p50 and p-p65 in the vaginal wall tissue of the POP group were higher than those of the control group.After knocking down EGR1 in fibroblasts,the expressions of p50 and p-p65 decreased,while the expression of p-IκBα increased.After overexpression of EGR1,the above molecules expressed the opposite level.Co-immunoprecipitation experiments found that EGR1 could target and regulate NFκB1.4.EGR1 is the downstream target gene of mi R-23a-3p,and mi R-23a-3p could inhibit the expression of EGR1.Overexpression of mi R-23a-3p could accelerate the proliferation of vaginal wall fibroblasts and reduce the proportion of apoptosis.Knockdown of mi R-23a-3p could slow down the proliferation of vaginal wall fibroblasts and increase the proportion of apoptosis.Conclusion: Differential endogenous expression of mi R-23a-3p and EGR1 was found in vaginal wall tissues of POP patients and control patients.In the POP group,the expression level of MMP9 in vaginal wall tissue increased,while the expression level of TIMP1 decreased,and the expression levels of collagen types I and III decreased,which may be the pathophysiological basis of POP.EGR1 can affect the proliferation,apoptosis and collagen metabolism of vaginal wall fibroblasts by targeting and regulating NFκB1 to affect the NFκB signaling pathway.EGR1 is the downstream target gene of mi R-23a-3p.Mi R-23a-3p can inhibit the expression of EGR1,thereby regulating the synthesis of collagen,and providing new ideas for the occurrence and development mechanism and preventive treatment of POP in the future. |
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