Research The Role Of Cofilin-1 In Pathogenesis Of Endometriosis

ObjectiveEndometiosis is a common and frequently encountered disease in child-bearing period of women. Although endometriosis is benign disease, endometriosis presents some characteristics of malignant tumor such as infiltration and metastasis. The pathogenesis of endometriosis has not been elucidated up to now. The theory of implantation of endometrial cells and fragments refluxed during the menstrual period is generally accepted in the pathogenesis of EMs. However this theory could not explain that the incidence rare of menstrual bleeding reflux was 80%-90% and the incidence rare of endometriosis is 10%-15%. A number of research suggested that abnormal genes expression in eutopic endometrium of endometriosis was probably the basis of endometriosis genesis and these abnormal genes expression probably have direct influence the pathogenesis of endometriosis. Cofilin-1(CFL1) gene was one of the differential expression genes between eutopic endometrium of endometriosis and normal endometrium screened by cDNA representative difference analysis (cDNA-RDA). There is not any report about the role of CFL in the pathogenesis of endometriosis.CFL1 gene locates in 11ql3. CFL1, a small (19 kD) and ubiquitous cytoskeletal protein, belongs to the actin-depolymerizing factor (ADF) family. CFL1 plays a vital role in promotion of actin depolymerization/poly-merization and rapid turnover of actin filaments. The reorganization of actin cytoskeleton is crucial in tumor progression, cell motility, cell adhesion, cell invasion, and angiogenesis. Inhibition of CFL1 activity in carcinoma cells depresses cell motility. Downregulation of cofilin expression reduces the assembly and stability of invadopodia, indicating a critical role of cofilin in cell invasion. Moreover, angiogenesis depends on the regulation of actin cystoskeleton dynamics by CFL1 and CFL1 has been identified as the target of a few of angiogenesis inhibitors.According to the theory of implantation of endometrial cells and fragments refluxed during the menstrual period adhesion, invasion and angiopoiesis was the elementary genesis process of endometriosis. Whether CFL1 which is related to adhesion, invasion and angiopopoiesis participates the genesis of endometriosis? What is the role of CFL1 in genesis of endometriosis? So in this study the role of CLF1 in genesis of endometriosis was investigated.Mehtods1,The expression of CFL1 in normal endometrium, eutopic endometrium and endometriotic lesions of endometriosis.(1) Real time fluorescence quantitive PCR was used to detect the expression of CFL1 mRNA in normal endometrium, eutopic endometrium and endometriotic lesions of endometriosis.(2) Western blot was used to detect the expression of CFL1 protein in normal endometrium, eutopic endometrium and endometriotic lesions of endometriosis.2,The effects of CFL1 on biological activity of eutopic endometrium stromal cells of endometriosis(ESC) and normal endometrium stromal cells (NSC).(1) Primary culture of ESC and NSC.(2) Identifieation of ESC and NSC by immunocytochemistry.(3) Three CFL1shRNA expression vector were transiently transfected to ESC to knockdown the CFL1 expression in ESC. (4) Molecular cloning technology was used to construct the recombinant pEGFP-N1-CFL expression vector of human CFL1 gene. The vector wasidentified with enzyme digestion and sequeneing.(5) Real time fluorescence quantitive PCR was used to detect the effect of RNA interference targeting CFL1 in ESC and the effect of transfection of recombinant pEGFP-Nl-CFL expression vector in NSC.(6) Western blot was used to detect the effect of RNA interference targeting CFL1 in ESC and the effect of transfection of recombinant pEGFP-Nl-CFL expression vector in NSC.(7) CCK-8 cell proliferation assay and growth curve were used to investigate the effect of CFL1 on the proliferation of ESC and NSC.(8) Annexin V-PE/7-AAD apoptosis detection kit was used to investigate the effect of CFL1 on the apoptosis of ESC and NSC.(9) Cell adhesion assay was used to investigate the effect of CFL1 on the adhesion capacity of ESC and NSC.(10)Transwell cell invasion assay was used to investigate the effect of CFL1 on the invasion capacity of ESC and NSC.(11) Enzyme linked immunosorbent assay(ELISA) was used to investigate the effect of CFL1 on the ICAM-1,VEGF and MMP-9 secretion of ESC and NSC.3,With vivo test to analyse the effect of expression of CFL1 on implanationability of ESC or NSC(1) Primary culture of ESC and NSC.(2) Identifieation of ESC and NSC by immunocytochemistry.(3) Three CFL1 shRNA expression vector were transiently transfected to ESC to knockdown the CFL1 expression in ESC.(4) Molecular cloning technology was used to construct the recombinant pEGFP-N1-CFL expression vector of human CFL1 gene. The vector wasidentified with enzyme digestion and sequeneing.(5) Construction of endometriosis nude mice model with abdominal injection method and calculate the volume of ectopic lesions.Results1,The expression of CFL1 in normal endometrium, eutopic endometrium and endometriotic lesions of endometriosis.(1) The results of real time fluorescence quantitive PCR showed that relative expression level of CFLl mRNA was significantly higher in eutopic endometrium (4.56±1.90) and endometriotic lesions of endometriosis(4.81±1.44), than that in normal endometrium(1.00±0.35).(2) The results of western blot showed that relative expression of CFLl protein level was notablely upregulated in eutopic endometrium(3.37±0.30) and endometriotic lesions of endometriosis(3.35±0.35)), compared with that in normal endometrium(1.00±0.39).2,The effects of CFL1 on biological activity of eutopic endometrium stromal cells of endometriosis(ESC) and normal endometrium stromal cells(NSC).(1) The primary culture of ESC and NSC was successfully proceeded.(2) The result of immunocytochemistry showed that staining of with antibodies against vimentin and CD10 were positive and staining with antibodies against cytokeratin, factor VIII and leukocyte common antigen was negative.(3) By the identification of sequeneing, the three CFL1 shRNA expression vectors and pEGFP-Nl-CFL recombinant plasmid were successfully established.(4) Real time PCR showed that CFL1 mRNA was overexpressed in ESC under basal condition. After transfection of three vectors, the expression of CFL1 mRNA was all decreased. Transfection of vector 2 (pRT-CFLl-s2) had the strongest effect on the knockdown of CFL1 mRNA level. The expression of CFL1 mRNA levels did not change after transfection of control plasmids (pRT-sNC or pRT-V). The expression of CFL1 mRNA was upregulated after transfection of pEGFP-Nl-CFL recombinant plasmid. The expression of CFL1 mRNA levels did not change after transfection of the control void plasmids (pEGFP-N1-V).(5) Western blot analysis showed that CFL1 protein was overexpressed in ESC under basal condition. After transfection of three vectors, the expression of CFL1 protein was all decreased. Transfection of vector 2 (pRT-CFLl-s2) had the strongest effect on the knockdown of CFL1 protein level. Therefore, vector 2 (pRT-CFL1-s2) was used for the further experiments. The expression of CFL1 protein levels did not change after transfection of control plasmids (pRT-sNC or pRT-V). The expression of CFL1 protein was upregulated after transfection of pEGFP-N1-CFL recombinant plasmid. The expression of CFL1 protein levels did not change after transfection of the control void plasmids (pEGFP-N1-V). Transfection of vector 2 (pRT-CFL1-s2) had the strongest effect on the knockdown of CFL1 Mrna and protein level. Therefore, vector 2 (pRT-CFL1-s2) was used for the further experiments.(6) The result of cell proliferation assay showed under basal condition, the proliferation of ESC was more vigorous than that of NSC. Silencing of CFL1 gene in ESC significantly decelerated the proliferation. The slope of proliferation curve after transfection of vector 2 (pRT-CFL1-s2) was almost as same as that of NSC, indicating that the proliferation rate of ESC had recovered to normal after silencing of CFL1 gene. CFL1 gene upregulating accelerated NSC proliferation. The proliferation and apoptosis rate of NSC did not change after transfection of of void pEGFP-N1 plasmid(7)The result of cell apoptosis assay showed under basal condition, the apoptosis rate of ESC was lower than that of NSC. After silencing of CFL1 gene, the apoptosis rate of ESC was accelerated. The proliferation and apoptosis rate did not change after transfection of control plasmids (pRT-sNC or pRT-V). CFL1 gene upregulating decelerated NSC apoptosis rate. The apoptosis rate of NSC did not change after transfection of of void pEGFP-N1 plasmid.(8) The result of cell adhesion assay showed under basal condition, adhesion of ESC was more intensive than that of NSC. After silencing of CFL1 gene, the adhesion of ESC was significantly reduced. The adhesion of ESC did not change after transfection of control plasmids (pRT-sNC and pRT-V). After upregulating of CFL1 gene, the adhesion of NSC was significantly enhanced. The adhesion of NSC did not change after transfection of of void pEGFP-N1 plasmid.(9) The result of cell invasion assay showed under basal condition, the invasion of ESC was more intensive than that of NSC. After silencing of CFL1 gene, the invasion of ESC was significantly depressed compared to baseline. The invasion of ESC did not change after transfection of control plasmids (pRT-sNC and pRT-V). After upregulating of CFL1 gene, the invasion of NSC was significantly enhanced compared to baseline (Figure 4D). The invasion of NSC did not change after transfection of of void pEGFP-N1 plasmid.(10) The result of ELISA assay showed under basal condition, ESC secreted more ICAM-1, MMP-9 and VEGF than NSC. After silencing of CFL1 gene, the secretion of ICAM-1, MMP-9 and VEGF from ESC were significantly inhibited compared to NSC. The secretion of ICAM-1, MMP-9 and VEGF from ESC did not change after transfection of control plasmids (pRT-sNC and pRT-V). After upregulating of CFL1 gene, secretion of ICAM-1, MMP-9 and VEGF from NSC was significantly enhanced. The secretion of ICAM-1, MMP-9 and VEGF from NSC did not change after transfection of of void pEGFP-N1 plasmid.(11) ESC had many prominences on the cell surface. The nucleus of ESC was irregular with much incision. Chromatin margination was not observed in ESC. After silencing of CFL1 gene, prominences on the surface of ESC was few and heterochromatin presented clumping and margination. NSC had few prominences on the surface. Chromatin margination was obviously observed in NSC. After upregulating of CFL1 gene, some prominences was observed on the surface of NSC without chromatin margination.3,The effects of CFL1 on vivo transplantation activity of eutopic endometrium stromal cells of endometriosis(ESC) and normal endometrium stromal cells(NSC).The volumes of endometriotic lesions derived from ESC were larger than that derived from NSC. In this model, extensive endometriotic lesions were detected in all nude mice. The vessels on the surface of the lesions were divided into many branches with tight and deep adhesion to the surrounding tissues. After silencing of CFL1 gene in ESC, the lesion volumes were reduced significantly. After silencing of CFL1 gene, the vessels on the surface of the lesions were less divided. The lesion volumes did not change after transfection of control plasmids (pRT-sNC and pRT-V). After upregulating of CFL1 gene, the lesion volumes were reduced significantly. The lesion volumes from NSC did not change after transfection of of void pEGFP-N1 plasmid.Conclusions1,CFL1 is found to express highly in eutopic endometrium and endometriotic lesions of endometriosis compared with normal endometrium.2,Overexpression of CFL1 was related to enhanced cell proliferation, reduced cell apoptosis, enhanced cell adhesion and invasion capacity, more secretion of ICAM-1,VEGF and MMP-9 of ESC. Differential expression of CFL1 between ESC and NSC was the important factor contributed to differential biological behaviours between ESC and NSC.3,Vivo transplantation capacity of ESC was more intensive than that of NSC. Overexpression of CFL1 is associated with enhanced vivo transplantation capacity of ESC. Differential expression of CFL1 between ESC and NSC was probably the important factor contributed to differential vivo transplantation capacity between ESC and NSC.