| Chapterl.The estrogen-regulated IncRNA H19/miR-216a-5p axis alters stromal cell invasion and migration via ACTA2 in endometriosis Background:Endometriosis(EMs)refers to the endometrium tissue(glands and stroma)appears outside the uterine body.It is an estrogen dependent disease.The clinical symptoms are chronic pelvic pain,sexual intercourse pain,irregular menstruation,infertility,etc.The incidence rate is about 10%in the women of childbearing age,as high as in infertile women.It is a common gynecological disease that affects the physical and mental health of women.Although endometriosis is benign in morphology,it is similar to malignant tumor in clinical behavior,such as ectopic implantation,growth,invasion and migration.Therefore,it is of great significance to study the pathogenesis of endometriosis."Eutopic endometriosis determinism" points out that the biological characteristics of endometrium is the decisive factor in the occurrence of endometriosis,and local microenvironment changes are the influencing factors.The clinical diagnosis of endometriosis is that ectopic lesions are found under laparoscopic operation.According to the pathophysiological characteristics of the disease,because of the periodic damage repair of the invasion and migration of the endometrium cells to the ectopic site,there are many fibrous connective tissue surrounding in the ectopic lesions.The stromal tissue has the ability to invade and migrate and the endoplasmic fibrosis changes to fibrosis.The high-level estrogen stimulation in vivo will lead to fibroblast-to-myofibroblast transdifferentiation,and accelerate the invasion and migration of endometrial cells.Thus,it causes a series of clinical symptoms such as pelvic pain and infertility.LncRNA H19 is the first discovered long non coding RNA.H19 can promote the proliferation,invasion and migration of tumor cells,and inhibit apoptosis in tumor cells,but lack of research in endometriosis.Since fibrotic encapsulation of ectopic lesions is an important marker of the occurrence,development and pathological grading of the endometriosis,ACTA2,as an important marker of fibrous tissue smooth muscle cells,has not been reported in eutopic endometrium of endometriosis.We intend to explore the role of H19 and ACTA2 in endometriosis and the pathogenesis of fibrotic encapsulation of ectopic lesions in order to find a new diagnosis target and treatment.Objective:To clarify the expression of H19 in endometriosis,and further study on potential functional effects and molecular mechanisms of H19 on downstream genes in eutopic endometrium,especially to explain the phenomenon of fibrous tissue and fibrosis surrounding in ectopic lesions,among which ACTA2 is an important marker of fibrous smooth muscle cells.We intend to explore the relationship between estrogen,H19,ACTA2 and its impact on eutopic endometrium.In order to explore the pathogenesis of endometriosis,and provide research basis for clinical diagnosis and treatments.Methods:In this chapter,we collected 23 cases of endometriosis eutopic endometrium as experimental group,and 23 cases of female endometrium as the control groupmatched.Instudy on gene expression,the expression levels of H19 and ACTTA2 were verified by qRT-PCR and WB on euESC,and the correlation was analyzed by Pearson test;in study of gene function,the effects of H19 and ACTA2 on the proliferation of ENDO euESC were studied by EdU and CCK8 experiments,and the invasion and migration of H19 and ACTA2 on ENDO euESC were studied by Transwell experiments;In order to further explore the role of ceRNA in ENDO euESC,RNA rescue experiment was used to verify the regulatory role of miRNA between H19 and ACTA2,and the effect and function of miRNA on ENDO euESC.Because of endometriosis is an estrogen-dependent disease,we studied whether estrogen can affect the function of euESC in endometriosis by regulating the molecular mechanism of ceRNA.Results:qRT-PCR and WB results showed that H19 and ACTA2 were highly expressed in ENDO euESC,and there was a positive correlation between them,but there was no significant correlation in the control group.After knocking down H19 and ACTA2 respectively,the invasion and migration function of ENDO euESC decreased significantly,but in the aspect of cell proliferation,knocking down H19 significantly inhibited the proliferation level of ENDO euESC,Knockdown of ACTA2 did not significantly change the proliferation level of euESC;bioinformatics analysis showed that mir-216a-5p and H19,ACTA2 had targeted binding sites,and luciferase experiment also verified that mir-216a-5p could bind to the exon of H19 and the 3’UTR noncoding region of ACTA2 through specific targeting sites.According to RNA rescue experiments,we also found that miR-216a-5p could inhibit the downstream ACTA2 gene expression and the upstream H19 gene expression through competitive binding,so as to further regulating invasion and migration level of ENDO euESC.According to the research of estrogen,we found that ENDO euESC treated with short-term estrogen,can promote the invasion and migration of EMs euESC byregulating H19/miR-216a-5p/ACTA2,but had little effect on cell proliferation.Conclusion:H19 and ACTA2 are highly expressed in ENDO euESC,and there is a positive correlation between them.Low expression of H19 and ACTA2 can inhibit the invasion and migration of ENDO euESC.They are negatively regulated by the targeted binding sites of miR-216a-5p,it has negative effects on the invasion and migration of ENDO euESC.Estrogen can also promote the invasion and migration of ENDO euESC by regulating H19/miR-216a-5p/ACTA2 signaling pathway.Chapter2.Study on role and mechanism of STING/IRF3/IFNbl signaling pathway in endometriosisBackground:Endometriosis is a common gynecological endocrine disease that seriously affects women’s physical and mental health.At present,there is no effective means to completely treat endometriosis.The etiology of endometriosis is complex,so the study of etiology and pathogenesis is of great significance for early warning,diagnosis and treatment intervention of endometriosis.Noticeably,the theory of "Eutopic endometriosis determinism " not only considers that the biological characteristics of endometrium are the decisive factors of endometriosis,but also the changes of local microenvironment.Therefore,in addition to the study on the characteristics of endometrium in the previous chapter,many scholars also found that the changes of the patient’s internal environment lead to ectopic lesions be able to change in addition to the uterine cavity.The important reasons for external placement planting,growth,invasion and migration,more and more studies have confirmed that endometriosis is a chronic inflammatory disease.The immune imbalance plays an important role in the occurrence and development of endometriosis.Its pathogenesis is related to the imbalance of immune function.It is generally believed that eutopic endometrium through adhesion,proliferation,invasion and migration,immune escape,neovascularization,induce chronic inflammation and form ectopic lesions.This is due to the immune dysfunction of endometriosis and the imbalance of immune microenvironment of ectopic lesions,which is manifested by the weakening of immune monitoring function and the cytotoxic effect of immune killer cells so that the ectopic endometrium could not be removed effectively.In clinical work,we also found that ectopic lesions are usually accompanied by inflammation and hyperemia through laparoscopic surgery.Patients with endometriosis show the characteristics of autoimmune diseases.Macrophages,inflammatory cytokines,growth factors and angiogenic substances in peritoneal fluid increase,which can promote the survival and proliferation of ectopic endometriunm and lead to local fibrous hyperplasia and adhesion.In our previous work,we found that STING gene was differentially expressed in eutopic endometrium and non eutopic endometrium by proteomics.STING(stimulator of interferon genes)a natural immune related protein,is a kind of intracellular DNA receptor protein,which can recognize endogenous double stranded DNA and phosphorylate the downstream protein signaling pathway of cytoplasmic TBK1 and IRF3,enter the nucleus and stimulate the release of interference b1 into the cytoplasm.Objective:To study the role of STING gene in the pathogenesis of endometriosis,and to further study the function and potential molecular mechanism of STING protein in endometriosis,so as to provide a new research basis for clinical diagnosis and treatment of endometriosis.Methods:In this chapter,we collected 15 cases of endometriosis eutopic endometrium as the experimental group,and 15 cases of matched female endometrium as matched control group.4 pairs of endometrium were detected for differentially expressed proteins by iTRAQ proteomics.After the preliminary screening of proteomics,STING was significant different,so we used the remaining 11 pairs of samples to verifySTING expression by WB and IHC experiments.In addition,we studied the effects of STING on the proliferation,invasion and migration of HESC,and the role of STING signaling pathway in stromal cells.Results:After preliminary proteomic screening of eutopic endometrium in endometriosis,it was found that immune related molecular proteins were important components,and the natural immune related STING were significantly different.According to WB results,STIING was significantly lower expressed in eutopic endometrium of endometriosis,while IHC results showed that STING was expressed in both stroma and glandular epithelium of normal endometrium,but in endometriosis,STING was mainly expressed in the stroma of eutopic endometrium,and mainly in glandular epithelium of ectopic endometrium.Further study on the role of STING on endometrial stromal cells showed that low expression of STING could promote theHESC proliferation,invasion and migration.The effect of STING/IRF3/IFNbl signaling pathway in HESCwith low expression of STING was also reduced,mainly showed the decreased expression of phosphorylated IRF3 and tbk1,and the decreased secretion of IFNb1.In order to further study the effect of IFNbl,secreted by STING/IRF3/IFNb1 signaling pathway,on stromal cells,we added exogenous IFNB1 to the HESC with low expression of STING,and found that IFNbl could reverse the invasion and migration function of stromal cells,but had little effect on cell proliferation.Conclusion:In this chapter,we found that STING with low expression and promoted cell invasion and migration in eutopic endometrium of endometriosis,also found that the expression and distribution of STING in endometriosis were significantly different,mainly in the stroma of eutopic endometrium and epithelium of ectopic endometrium.Meanwhile,we clarified that STING could play an important role in stromal cells invasion and migration through STING/IRF3/IFNb1 signaling pathway.It further enriched the etiology research of natural immunity related endometriosis,and brought new ideas for further expanding the pathogenesis research of endometriosis and guiding clinical treatment. |
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