Study On The Pathogenesis Of FKBP52 In Endometriosis

Endometriosis(Ems)is a common gynecological disease affecting 10% of reproductive-aged women,leading to inflammation,pelvic pain,and infertility.EMS is characterized by the development of endometrial tissues(glands and stroma)outside the uterine cavity.It has the biological behavior of invasion and metastasis similar to tumor.The pathogenesis of EMs is complicated,the factors are interrelated,the exact cause is not clear,which is a hot and difficult point in clinical research.Regardless of the etiology of Ems,it has been widely recognized as a sex hormone-dependent disease.A high level of estrogen and/or low progesterone response is thought to be related to Ems,but their mechanisms are unclear.A growing number of studies have shown that low progesterone response is an important feature of Ems.Progestogen functions mainly through its nuclear receptor in the FKBP52 and Hsp90 and other molecular chaperones in the form of complex transport of progesterone into the nucleus to complete the transcription process and then activate the progesterone function.Previous studies have suggested that progesterone resistance in endometriosis appears to be associated with a decrease in progesterone response in endometrial stromal cells due to a decrease in PR expression.But so far,PRs have been implicated in the pathogenesis of Ems.FKBP52 is a subfamily of the FK506-binding protein(FKBP)family.As a chaperone of PRs,FKBP52 plays a key role in thematuration of PRs and the physiological function of P4.It is a kind of binding Hsp90 and PR to stabilize the complex to achieve the best binding of P4 to PR and subsequent transcriptional activation.Thus,the normal physiological function of P4 can be brought into play.Some scholars have reported that the lack of FKBP52 promotes the pathogenesis of Ems,but there are few related studies,the specific mechanism needs to be studied.Our previous study has already found that the low expression of FKBP52 caused the dysfunction of the PR complex,resulting in the embryonic lethality.Therefore,we speculate that FKBP52 may induce progesterone resistance by affecting PR and promote the pathogenesis of Ems.Recent studies have shown that the occurrence,development and metastasis of various gynecological diseases and even tumors are closely related to intracellular signaling pathways.The phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway plays an important role in cell growth,proliferation,differentiation,and protein synthesis This pathway is activated in Ems,but the mechanism is unclear.Akt among the overexpression of endometrial stromal cells may weaken decidualization through its downstream target FOXO1 and influence PR to promote the survival of endometrial ectopic cells,which effects of PR on the survival of endometrial ectopic cells.Therefore,the series dialogue mechanism between PR and Akt becomes the key point of mechanism research.There are many studies on hormone receptors in Ems at home and abroad,but there are few studies on the regulatory mechanism of hormone receptors.The purpose of this study was to study the pathogenesis of FKBP52 and Ems in clinical tissue samples and cytology,To elucidate the effect of FKPB52 gene function on the biological behavior of endometrial ectopic cells,and the mechanism of FKBP52and PR and PI3K/Akt/mTOR signaling pathwayThe research includes the following three parts:Part 1 Relationship between FKBP52,PR,PI3K/Akt/mTOR signaling pathway in Ems tissueObjective To determine whether a correlation existed among FKBP52,progesterone receptor(PR),phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway,and endometriosis(Ems)in clinical tissuesMethod15 were ectopic ones collected from patients with Ems(case group),and 15 were normal ones collected from infertile women without Ems(control group).The FKBP52 and PR m RNA expression levels in endometrial tissues were measured by quantitative real-time PCR.Protein levels of FKBP52,PR,HSP90,FOXO1,p-PI3 K,p-Akt,and p-mTOR were detected by Western blot analysis.Result1.Compared with the control group,the expression levels of FKBP52 and PR(PR-A and PR-B)m RNA were down-regulated in the case group,there was significant difference among FKBP52(P<0.05),but there was no significant difference among PR(PR-A and PR-B)(P>0.05).2.The expression levels of FKBP52,PR(PR-A,PR-B),HSP90,FOXO1 protein in the case group were significantly lower than those in the control group(P<0.05),whereas p-PI3 K,p-AKT,and p-mTOR were up-regulated(P<0.05).3.The expression levels of p-PI3 K,p-AKT,and p-mTOR protein in the case group were significantly up-regulated than those in the control group(P<0.05).Conclution Among Ems,FKBP52 was associated with the PR and PI3K/Akt/mTOR signaling pathway.The decrease in FKBP52 may be due to the abnormal expression of PR,resulting in the dysfunction of the PR complex and the occurrence of progesterone resistance,which affected FOXO1 and activated the Akt pathway and promoted the survival and proliferation of heterotopic cells.Part 2 Establishment of endometrial stromal cell model and bioinformatics analysis of FKBP52Objective A model of endometrial stromal cells was established and its characteristics were preliminarily analyzed to provide a reliable model for the study in vitro.Bioinformatics is used to analyze the gene characteristics of FKBP52,which provides a theoretical basis for the study.Method Ectopic endometrial tissue was obtained under laparoscope and primary culture of ectopic endometrial stromal cells(ESCs)was performed.Normal endometrial tissue was obtained by conventional curettage and primary culture of normal endometrial stromal cells(NSCs)was performed.CCK8 assay and flow cytometry were used to detect the proliferation and growth ability and apoptosis of ESC and NSC at 24 h,48h,72 h and 96 h respectively.The gene structure,protein structure,subcellular localization and expression pattern ofFKBP52 were analyzed by bioinformatics database.Result1.The cell model was successfully established by improving the method of primary stromal cell culture.2.The results of CCK8 method showed that the proliferation of NSC was significantly higher than that of NSC.According to the trend of proliferation curve,there was significant difference in the relative proliferation rate of ESC compared with NSC for 72 h.There was no significant difference in relative proliferation rate between the two groups(P<0.05)at 24 h and 96h(P>0.05).3.The results of flow cytometry showed that the apoptosis rate of the two groups was significantly lower than that of NSC.The apoptosis rate of the two groups was significantly lower than that of NSC for 48 h and 96 h.There was no significant difference in apoptosis rate between the two groups(P>0.05).4.The FKBP52 gene is located on human chromosome 12.The total length of protein is 459 amino acids.The expression of FKBP52 is different in different tissues.FKBP52 is located in the cytoplasm.FKBP52 binds to a variety of interacting proteins to form a stable complex structure and function.Conclution The improved primary culture method of endometrial stromal cells can effectively improve the success rate of primary cell culture.The ESC cell model has the disease characteristics of Ems.The crystal structure of FK2 and TPR regions in FKBP52 makes it play an important role in the regulation of steroid hormones.In particular,the proline rich loop loop structure,which covers the field of FKBP52 FK1 catalysis,is functionally important.Targeted therapy for proline circulatory interaction in FKBP52 is the most attractive therapy that interferes with the regulation of hormone receptor-dependent physiology anddisease receptor activity by FKBP52.FKBP52 is located in cytoplasm and it is feasible to study gene function in cell model.Part 3 Gene function of FKBP52 in Ems and its Mechanism of progesterone ResistanceObjective To study the gene function of FKBP52 in Ems at the cytological level and to determine whether FKBP52 is associated with the pathogenesis of Ems and how the specific mechanism is.Method FKBP52 overexpressed plasmids,unloaded,FKBP52-siRNA and NC-siRAN were transfected into human endometriosis stromal cells by lipofectamine2000 cationic liposome method.Divided into five groups:1.Blank control group;2.FKBP52 overexpression empty vector group(pcDNA 3.1);3.FKBP52 overexpression group(pcDNA 3.1-FKBP52);4.FKBP52 silencing group(siRNA-FKBP52);5.Blank control group of siRNA(siRNA-NC)?CCK8and flow cytometry were used to detect the biological behavior of cells.The expression level of FKBP52?PR?Akt m RNA were detected by qRT-PCR.The expression level of FKBP52,PR,HSP90,FOXO1,Akt,p-Akt,mTOR,p-Mtor protein were detected by Wb.Result1.Effect of FKBP52 Gene function on Cell Biological behavior(1)The results of CCK8 method: from the trend of proliferation curve,the proliferation of pcDNA 3.1-FKBP52 cells decreased and the proliferation of siRNA-FKBP52 cells increased.(2)The results of flow cytometry showed that the apoptosis rate of siRNA-FKBP52 cells was significantly higher than that of Blank cells(P<0.001)and that of siRNA-FKBP52 cells(P>0.05).2.Effect of FKBP52 Gene function on genes and proteins(1)The m RNA and protein of FKBP52 in the FKBP52 of FKBP523.1-FKBP52 were significantly higher than those of the Blankopsis cDNA3.1-FKBP52(P<0.001).The m RNA and protein of PR-A increased significantly(P<0.001).The m RNA and protein of PR-B decreased significantly(P<0.05).The m RNA and protein of Akt increased,but there was no significant difference(P>0.05).The expression of p-Akt protein increased significantly(P<0.05),indicating that the main activation of Akt pathway was due to the activation of phosphorylated Akt.The protein expression of m TOR and p-mTOR was significantly up-regulated(P<0.05).There was no significant difference in HSP90 protein(P>0.05).(2)Compared with BlanktsiRNA-FKBP52,the m RNA and m RNA of FKBP52 decreased significantly(P<0.001).The m RNA and protein of PR-A decreased significantly(P<0.001).The m RNA of PR-B decreased significantly(P<0.001)and the protein decreased,but there was no significant difference(P>0.05).The m RNA and the protein of Akt,the protein of p-Akt were decreased(p<0.05).The protein expression of m TOR and p-mTOR decreased significantly(P<0.05).There was no significant difference in HSP90 protein(P>0.05).ConclutionFKBP52 is an important pathogenic factor for Ems The function of FKBP52gene in Ems affects the biological behavior of endometriosis stromal cells.The mechanism of Ems progesterone resistance is that the low expression of FKBP52 gene leads to the imbalance of PR-A:PR-B.The abnormal PR signal decreased the ability of progesterone to counteract estrogen induced endometrial cell proliferation and/or response to pro-inflammatory stimulation,and promoted the growth of endometrial cells transferred to the peritoneum.The abnormal mechanism of Akt pathway in Ems is related to PR-A,but the specific regulation is still not clear.The further study on the function of FKBP52 gene in Ems may provide a new idea and new target for the treatment of endometriosis.