Effects Of RhoA/ROCK Signal Pathway Induced By PRL-2on Regulating Edometriotic Cell Migration

Background and Objective:Endometriosis is a common benign gynecological disorder affecting women's physical and mental health, quality of life and even fertility. Several theories related with endometriosis have been proposed to explain the etiology of endometriosis, but the exact pathogenesis and related infertility remain unclear. For example, how do the endometrial cells migrate from the uterine cavity to ectopic location through a series of complex processes including adhesion, invasion and angiogenesis. Furthermore, how do these cells process the signals resulting in its migration and the key regulators involved in this process are required to be elucidated. So it is imperative to reveal the mechanisms involved in the migration of endometriotic cells.Phosphatase of regenerating liver-3(PRL-3) has recently been a focus in cancer research as a promising prognostic factor and therapeutic target in metastatic tumors for it s important role in the cancer cell metastasis process. Since Saha found PRL-3had close relationship with colon cancer metastasis in2001. more and more research have shown that PRL-3high expression seEMs to be a common event in the development of a variety of tumors especially in cancer cell metastasis. PRL-3can activate a series of intracellular signal transduction pathway, inducing cell proliferation.invasion and metastasis to improve its ability to induce aggression, adhension, angiogenesis. Recent studies show that PRL-3as an upstream signal induce its downstream Rho/ROCK signaling pathway to promote tumor development, ultimately leading to the change of cell proliferation and migration capabilities. Studies shew that RhoA and ROCK expressed high level in ectopic endometrial stromal cells, however there is no research on the relationship between PRL-3and endometriosis. Also, many studies have found endometriosis shares similar biological phenotype with malignant tumors in excessive proliferation, metastasis and invasion.Based on the above research background, we designed this project. Firstly we compared PRL-3protein expression in ectopic, eutopic endometrium and non-EMs endometrium by immunohistochemistry and Western Blot technology to explore the correlationship between PRL-3expression and endometriosis. Secondly though the primary culture of endometrial stromal cells, we compared cell migration ability and PRL-3, T-RhoA, P-RhoA and ROCK gene expression among ectopic, eutopic and non-EMs endometrium. Thirdly we used PRL-3siRNA to silence PRL-3expression, then real-time RT-PCR and Western Blot methods were to detect PRL-3, RhoA and ROCK gene expression on cellular and molecular level. Then laser scanning confocal microscopy, scratch test and transwell cell migration experiments were used to explore the changes of transfected ectopic endometrial stromal cells cytoskeleton and migratory ability.The present study aimed to investigate how PRL-3mediate RhoA/ROCK signaling pathway on the regulating endometriotic cell migration and its molecular mechanism in-depth, which will further reveal the pathogenesis of EMs and provide a new way of its treatment. Object:To explore the relationship between abnormal expression of PRL-3and endometriosis.Material and Methods:On one hand,70ectopic and eutopic endometrial samples from EMs patients and30non-EMs endometrial samples were obtained for PRL-3immunohistochemical staining analysis, all patients were followed-up for3years. On the other hand, PRL-3protein expression was detected by Western Blot method in35ectopic and eutopic endometrial frozen tissue from EMs patients and20non-EMs endometrial frozen tissue. In order to compare the differences of PRL-3protein expression in ectopic, eutopic and non-EMs endometrial tissue.Results:Immunohistochemical staining shew PRL-3protein was expressed in the cytoplasm of ectopic endometrial cell. Moreover, the expression of PRL-3protein in ectopic tissue was significantly higher than thoses of eutopic and non-EMs endometrial tissues, and statistic analysis indicated the expression of PRL-3was closely associated with the clinical stage and recurrence of endometriosis (P＜0.05).Western Blot also shew the expression of PRL-3protein in ectopic endometrial tissues was much higher than those of eutopic and non-EMs endometrial tissues (P＜0.001)Conclusion:The expression of PRL-3in ectopic endometrial tissues was much higher than those of eutopic and non-EMs endometrial tissues, moreover its expression had close relationship with clinical stage and recurrence of endometriosis. Object:To compare cell migration ability and PRL-3/RhoA/ROCK signal pathway expression in ectopic,eutopic and non-EMs endometrial stromal cells in vitro.Material and Methods:In order to establish cell migration model, ectopic. eutopic and non-EMs endometrial stromal cells were primary cultured in vitro,which were identified by vimentin staining. Cell migration ability was detected by cell pseudopod formation, scratch test and transwell experiments in three groups. Then PRL-3, T-RhoA, P-RhoA and ROCK gene expressions were analysed by western blot and real-time RT-PCR.Results:Transwell experiments showed the migration stromal cells through matrigel of ectopic, eutopic and non-EMs groups were4.13±0.81,2.65±1.07and1.45±0.39respectively in the same time. There were significant differences among three groups (P <0.05). The migration cells in ectopic group were more than that of eutopic group(P <0.05). moreover, there was significant differences between eutopic and non-EMs group (P<0.05). Scratch experiments also showed cell migration ability was much faster in ectopic group, while medium in eutopic group, and slowest in non-EMs group. PRL-3protein expression was0.245±0.132in ectopic group, while it was not detected in eutopic and non-EMs groups, so there were significant differences amoung them (P<0.01). Also PRL-3, T-RhoA, P-RhoA and ROCK gene expressions were found much stronger in ectopic group, while medium in eutopic group, and slowest in non-EMs group by western blot and real-time RT-PCR analysis.Conclusion:1. cell migration ability:ectopic group> eutopic group>non-EMs endometrial group.2. PRL-3/RhoA/ROCK signal pathway:ectopic group> eutopic group>non-EMs group. Object:To deeply explore the effects of RhoA/ROCK signaling pathway induced by PRL-3on regulating endometriotic cell migration and its molecular mechanism by the interference of PRL-3siRNA.Material and Methods:Primary endometriotic stromal cells were cultured in vitro, PRL-3siRNA was transfected to silence PRL-3gene, then PRL-3, T-RhoA, P-RhoA and ROCK gene expression was analysed by western Blot and real-time RT-PCR. The changes of cell migration was detected by cell pseudopod formation, scratch test and transwell cell migration test. The changes of endometriotic stromal cells cytoskeleton was observed by laser scanning confocal microscopy.Results:After transfection72h, PRL-3protein expression was respectively0.049±0.052,0.364±0.128and0.418±0.090in experimental group, negative control group and blank control group. PRL-3protein expression had statistically differences among the three groups (P<0.05), while there was no difference between negative control group and blank control group (P>0.05). The expressions of PRL-3, T-RhoA, P-RhoA and ROCK were decreased a lot in endometriotic stromal cell during PRL-3siRNA transfection0- 72h. Especially from24h to48h. this inhibition effect is more rapid and obvious for PRL-3, its inhibition rate was about81.4%up to72h. The variation tendency of P-RhoA expression was accordance with PRL-3during siRNA transfection0-72h. The formation of cell pseudopod in experimental group was less than that of control groups. Within the same time, the number of migration cells in experimental group was0.87±0.21which was less than those of1.75±0.28in blank control group and1.63±0.39in negative control group (P＜0.05). Stress fibers became thicker and clearer in the experimental group, while filaments and pseudopodia plate significantly reduced compared with control group.Conclusion:1. PRL-3siRNA could effectively silence PRL-3gene, and down-regulate RhoA/ROCK signal pathway;2. Endometriotic stromal cells migration ability decresed a lot after transfected with PRL-3siRNA;3. PRL-3regulated endometriotic cell migration through RhoA/ROCK signal pathway.