Studies Of The Effect And Mechanism Of Periostin And Emodin On The Migrtion And Invasion Abilities Of Endometriosis By Targeting ILK

BackgroundEndometriosis is a benign disease which affects almost 10%-15% of women in their reproductive period. Besides to dysmenorrhea and chronic pelvic pain, endometriosis can also result in dyspareunia, infertility and and a series of clinical symptoms. Although endometriosis is a benign disease, it exhibits malignant-like biological behaviors, namely, adhesion, implantation, invasion as well as angiogenesis. Up to date, there is no radical cure for endometriosis due its unclear pathogenesis. Thus, it is of great significance for improving the prognosis of endometriosis to go deeply into discussing the relationship between fuction changes of new genes and the pathogenesis of endometriosis, to explain its mechanism from the molecular level, as well as to seek for an effective targeted therapy for endometriosis.Periostin belongs to Matricellular Proteins family, which is a kind of extracellular matrix molecules in mediating the interaction between cells and extracellular matrix (ECM), and facilitates the survival, ashesion and invasion of cells. In structure, Periostin has an N-terminal, a cysteine-rich domain (EMI), four internal homologous repeats (FAS domain) and a C-terminal, of which the FAS1 domain contains binding site of integrins. Periostin can bind to integrins and activate the integrin pathway to modulate the cell abilities of survival, adhesion and migration. It is reported that periostin is highly expressed in a variety of malignant cancers and can promote the distant metastasis of cancers.Emodin is a naturally occurring anthraquinone presenting in the roots and barks of numerous plants, such as rhubarb, radix polygoni multiflori, giant knotweed and cassia seed. As an active ingredient of various Chinese herbs, emodin possesses various biological activities, including antibacterial, anti-inflammatory, anticancer and immunosuppressive effects. Recently, it is pointed out that emodin plays an important role in promoting the apoptosis and inhibiting the proliferation, invasion and metastasis of cancer cells. Relevant report points out that emodin downregulated the expression of transcription factors of integrin-linked kinase (ILK), Spl and c-Jun, resulted in the downregulation of the expression of ILK and supression of cell growth.Integrin-linked kinase (ILK), weight of 59 KD, plays essential roles in mediating the relationship between cell-ECM and cell-cell interaction. Due to its serine-threonine kinase activity, ILK can directly phosphorylate several downstream targets, such as GSK-3β, Akt, Erkl/2 and NF-κB, to mediate several signal pathways and modulate the survival, proliferation, migration and invasion of cells. Additionally, ILK can facilitate the epithelial-mesenchymal transition (EMT) of cells by up-regulating the EMT related transcription factors. ILK is highly expressed in a wide variety of cancers and is associated with cancer metastasis and prognosis, while ILK silencing inhibited the EMT, migration and invasion of cells. However, research about ILK was mostly concentrated in malignant tumor, the expression and function of ILK in endometriosis has not been described.Part I The study of the effect and mechanism of Periostin on the migrtion and invasion abilities of endometrial stromal cells by targeting ILKObjective:1. Detect the expression of Periostin in eutopic and ectopic endometrial stromal cells of patients with endometriosis.2. Explore the effect of Periostin on the proliferation, adhesion, migration and invasion abilities of endometrial stromal cells and the study of related mechanism.Methods:Endometrial stromal cells (ESCs), including control endometrial stromal cells (CSCs), eutopic and ectopic endometrial stromal cells (EuSCs and EcSCs), were isolated and cultured from patients with and without endometriosis. Real-time PCR and western blot were used to detect the expression of Periostin in EuSCs and EcSCs from patients with endometriosis. Western blot was used to detect the expression of integrin-linked kinase (ILK) and p-Akt in CSCs, EuSCs and EcSCs. The migration and invasion ablilities of CSCs, EuSCs and EcSCs were examined by transwell assays. Real-time PCR and western blot were used to detect the transfection effect after silencing the expression of Periostin in EcSCs using the small interfering RNA (siRNA). After EcSCs receiving the siRNA-Periostin, the levels of ILK and p-Akt were detected by western blot, abilities of proliferation, adhesion, migration as well as invasion of EcSCs were evaluated by the MTT assay, cell adhesion assay and transwell assays.Results:1. The identification and purity of ESCs:Human ESCs were isolated and cultured, and the purity of ESCs in our study was 96.5%±2.5%;2. Expression of Periostin, ILK and p-Akt in ESCs:The protein and mRNA levels of Periostin were up-regulated in EuSCs and EcSCs compared with CSCs, and the highest in EcSCs (protein:EuSCs vs CSCs P< 0.05, EcSCs vs CSCs P< 0.001; mRNA:EuSCs vs CSCs P> 0.05, EcSCs vs CSCs P< 0.05). No significant difference was found in the expression of Periostin in the proliferative and secretory phase (P> 0.05). The expression of ILK and p-Akt were enhancedand in EuSCs and EcSCs compared with CSCs (P< 0.05), and no significant difference existed in EuSCs and EcSCs (P> 0.05).3. Effect of Periostin on the expression of ILK and p-Akt in ESCs:The expression of Periostin was significantly downregulated after transfected EcSCs with siRNA-Periostin (P< 0.05). The expression of ILK and p-Akt were also downregulated after Periostin siRNA interference (P< 0.05).4. Effect of Periostin on the proliferation and adhesion abilities of ESCs:The proliferation ability of EuSCs and EcSCs were stronger than CSCs, but there was no significant difference (P> 0.05). The proliferation ability didn’t change significantly after Periostin siRNA interference (P> 0.05). The adhesion ability of EcSCs was stronger than CSCs (P< 0.05). No significant difference existed in the adhesion ability of EuSCs and CSCs (P> 0.05). The adhesion ability of EcSCs was decreased significantly after Periostin siRNA interference (P<0.05).5. Effect of Periostin on the migration and invasion abilities of ESCs:The migration and invasion abilities of EuSCs and EcSCs were stronger than CSCs, and the strongest in EcSCs (migration:P< 0.01; invasion:P< 0.001). The migration and invasion abilities of EcSCs were decreased significantly after Periostin siRNA interference (migration:P< 0.05; invasion:P< 0.01).Conclusion:1. Periostin enhances the abilities of adhesion, migration and invasion of ESCs, but has no effect on the proliferatin ability of ESCs.2. Periostin facilitates the development and progression of endometriosis by enhancing the adhesion, migration and invasion abilities of ESCs via the ILK signal pathway.Part II The study of the effect and mechanism of Periostin on the epithelial to mesenchymal transition of endometrial epithelial cells via targeting ILKObjective:1. Explore the effect of Periostin on the migration and invasion abilities of endometrial epithelial cells (EECs).2. Explore the effect of Periostin on the epithelial to mesenchymal transition (EMT) of endometrial epithelial cells and the study of related mechanism.Methods:EECs were isolated and cultured from patients with endometriosis. Transwell assays were used to evaluate abilities of migration and invasion of EECs before and after the treatment of Periostin. Western blot was used to detect the expression of integrin-linked kinase (ILK), E-cadherin, keratin, N-cadherin, vimentin, p-Akt, Slug and Zebl in EECs before and after the treatment of Periostin. After receiving ILK small interfering RNA (siRNA-ILK), western blot was also used to detect the expression of ILK, E-cadherin, keratin, N-cadherin, vimentin, p-Akt, Slug and Zebl in EECs with and without the treatment of Periostin.Results:1. The identification and purity of EECs:Human EECs were isolated and cultured, and the purity of EECs in our study was 95.6%±3.5%.2. Effect of Periostin on the migration and invasion abilities of EECs:After treated with periostin (20ng/ml and 40ng/ml) for 48 h, the migration and invasion abilities of EECs were enhanced significantly (migration:20ng/ml P< 0.01,40ng/ml P<0.001; invasion:20ng/ml P< 0.05,40ng/ml P< 0.01).3. Effect of Periostin on the expression of EMT-related protein, ILK and p-Akt in EECs:After treated with periostin for 48 h, the levels of E-cadherin and keratin were decreased in EECs, while the expression of N-cadherin and vimentin were markedly increased in the EECs, especially for the 40ng/ml group (P< 0.05). Furthermore, the expression of ILK, p-Akt, slug and Zebl were also markedly up-regulated by periostin in EECs, especially for the 40ng/ml group (P< 0.05).4. Effect of ILK silencing on the expression of ILK, p-Akt and EMT-related protein in EECs:After transfected with the siRNA-ILK, the expression of ILK, p-Akt, slug and Zebl were all down-regulated significantly in EECs (P< 0.05). Additionally, E-cadherin and keratin were up-regulated (P< 0.05) while N-cadherin and vimentin were down-regulated (P< 0.01) in EECs after receiving ILK silencing. Although above effects were weakened by periostin (40ng/ml), periostin failed to change the expression of p-Akt, slug, Zebl and EMT-related protein in EECs receiving ILK silencing when compared to normal EECs or EECs receiving blank siRNA sequence (P> 0.05). Thus, periostin failed to upregulate the expression of p-Akt, slug and Zebl as well as to facilitate the EMT of EECs after receiving ILK silencing.Conclusion:1. Periostin enhances the migration and invasion abilities of EECs.2. Periostin can induce the EMT of EECs.3. Periostin enhanced the EMT of EECs via the ILK signal pathway.Part Ⅲ The study of the effect and mechanism of emodin inhibites the migration and invasion abilities of endometrial stromal cells through facilitatingthe mesenchymal to epithelial transition of cells via targeting ILKObjective:1. Detect the expression of integrin-linked kinase (ILK) and migration and invasion abilities of eutopic and ectopic endometrial stromal cells (EuSCs and EcSCs) from patients with endometriosis.2. Explore the effect of emodin on the proliferation, migration and invasion abilities of endometrial stromal cells (ESCs).3. Explore the effect and mechanism of emodin on the mesenchymal to epithelial transition (MET) of ESCs.Methods:ESCs, including control endometrial stromal cells (CSCs), EuSCs and EcSCs, were isolated and cultured from patients with and without endometriosis. Western blot was used to detect the protein expression of ILK in CSCs, EuSCs and EcSCs. The migration and invasion abilities of CSCs, EuSCs and EcSCs were evaluated by the transwell assays. After treated with increasing concentratins of emodin, the proliferation, migration and invasion abilities of EcSCs were detected by MTT assay and transwell assays. Western blot was used to detect the expression of ILK, E-cadherin, keratin, N-cadherin and vimentin in EcSCs after treated with increasing concentratins of emodin. After EcSCs receiving ILK small interfering RNA (siRNA) as well as CSCs receiving pEGFP-C1-ILK, western blot was used to detect the protein expression of ILK, E-cadherin, keratin, N-cadherin and vimentin in EcSCs. Transwell assays were used to evaluate migration and invasion abilities of ESCs before and after the transfection. All above effect were detected after treatment with emodin in cells before and after the trasfection.Results:1. The expression of ILK and abilities of migration and invasion in ESCs:The expression of ILK was higher in EuSCs and EcSCs compared with that in CSCs, and highest in EcSCs (EuSCs vs CSCs:P< 0.01; EcSCs vs CSCs:P< 0.001). EuSCs and EcSCs were larger and more spindle-like than CSCs in morphology. The migration and invasion abilities of EuSCs and EcSCs were stronger than CSCs, and the strongest in EcSCs (migration:P< 0.01; invasion:EuSCs vs CSCs P< 0.05, EcSCs vs CSCs P< 0.01).2. Effect of emodin on the proliferation, migration and invasion abilities of ESCs: Emodin inhibited the proliferation of EuSCs and EcSCs in a dose-and time-dependent manner. EcSCs were smaller and less spindle-like after the treatment with emodin. The migration and invasion abilities of EcSCs were significantly decreased after treatment with emodin (migration:20μM P> 0.05,40μM P< 0.01; invasion:20μM P< 0.05,40μM P< 0.01).3. Effect of emodin on the expression of ILK and EMT-related protein in EcSCs: The levels of ILK, N-cadherin and vimentin were decreased, while the expression of E-cadherin and keratin were increased significantly in EcSCs after treated with increasing concentrations of emodin (P< 0.05). Moreover, the levels of ILK, N-cadherin and vimentin were also decreased, while the expression of E-cadherin and keratin were increased significantly in EcSCs after treated with emodin (40μM) with increasing treatment time (P< 0.05).4. Effect of ILK silencing on the expression of proteins, migration and invasion abilities of EcSCs:We silenced the ILK in EcSCs by transfecting EcSCs with siRNA-ILK because EcSCs showed the highest level of ILK. Compared with EcSCs treated with N.C., the levels of ILK, N-cadherin and vimentin were decreased while the levels of E-cadherin and keratin were increased in EcSCs after transfection with siRNA-ILK (P< 0.001). In addition, the migration and invasion abilities of EcSCs were decreased significantly by the transfection of siRNA-ILK (migration:P< 0.01, invasion:P< 0.001). All above effect can be strengthened by emodin (P< 0.05).5. Effect of ILK overexpression on the expression of proteins, migration and invasion abilities of CSCs:ILK was exogenous expressed in CSCs by transfecting CSCs with pEGFP-C1-ILK because CSCs showed the lowest level of ILK. Compared with CSCs receiving pEGFP-C1, the levels of ILK, N-cadherin and vimentin were increased while the levels of E-cadherin and keratin were decreased after transfecting CSCs with pEGFP-C1-ILK (P< 0.05). Simultaneously, the migration and invasion abilities of CSCs were increased signicifantly by the transfection of pEGFP-C1-ILK (migration:P< 0.01, invasion:P<0.001). Although emodin abrogated the above effect, it failed to facilitate the MET and to inhibite the migratin and invasion abilities of CSCs after exogenous expression of ILK in CSCs.Conclusion:1. Emodin inhibited the proliferation, migration and invasion abilities of ESCs in a dose- and time-dependent manner.2. Emodin inhibited the migration and invasion abilities of ESCs by facilitating the MET of ESCs.3. Emodin inhibited the migration and invasion abilities of ESCs by facilitating the MET of ESCs via ILK pathway.