The Role Of Estrogen Non-classical Pathway Mediated By GPR30in The Nathogenesis Of Adenomyosis

Adenomyosis, characterized by the invasion and growth of functional endometrial glands and stroma deep within the myometrium of the uterus, usually occurs in patients with menorrhagia and dysmenorrhea in their reproductive years. In the process of basal endometrium penetrating into the inner myometrium, altered apoptosis and proliferation of eutopic endometrium possibly elucidate some aspects of the pathophysiology of adenomyosis. Although the etiology and pathophysiology remain unclear, adenomyosis has always been defined as an estrogen-dependent disease, and the high estrogen environment has been involved in the pathogenesis of adenomyosis. The classical mechanism of estrogen responsiveness acts through nuclear estrogen receptors (nER, ERa and ERpβ), which are reasonably well understood. It has been debated that the existence and function of plasma membrane estrogen receptor (mER).Recently, accumulating reports suggest that a novel7-transmembrane G protein-coupled estrogen receptor (GPR30or GPER-1) can regulate estrogen responsiveness in many estrogen-dependent pathologic diseases, such as breast cancer, endometriosis, and endometrial cancers. GPR30is highly expressed and17β-E2positively regulates cell proliferation and invasion potential in endometrial carcinoma and cancer cell lines. Also, overexpressed GPR30levels increase with advanced-stage in uterine carcinosarcoma. A microarray analysis has proved that estrogen and4-hydroxytamoxifen are shown to induce proliferation and migration through GPR30-mediated rapid non-genome effects in ERs-negative breast cancer cell. All these findings strongly indicate the existence and function of GPR30as a plasma membrane estrogen-binding receptor. And To our knowledge, there have been few published studies of GPR30distribution and expression in uterus with adenomyosis.In the present study, we verified that the role of GPR30on the cell proliferation, invasiveness and apoptosis in the eutopic endometrial cell in the patients with adenomyosis. First, there was significantly higher protein and mRNA level of GPR30in the eutopic and ectopic endometrium from patients with adenomyosis, compared with controls. In order to conduct GPR30RNAi, we constructed lentiviral-vector mediated shRNA sequence targeting human GPR30, then transfected the lentiviral-vector into the endometrial stroma cell. Then we established the optimal infection conditions and screened effective shRNA against GPR30through PCR and qPCR analysis. Finally, we observed the cell proliferation, invasiveness and apoptosis after GPR30RNAi, compared with negative shRNA in the same cell. After GPR30RNAi in the stroma cell, GPR30and downstream pAkt/Akt protein significantly decreased, and cell proliferation and invasiveness significantly inhibited. There were up-regulation of caspase-3active/pro ratio and bcl-2protein, and down-regulation of bcl-x protein after GPR30RNAi. Objective:To evaluate the distributions and expressions of GPR30, a new membrane estrogen receptor, in the eutopic and ectopic endometrium from patients with, and without adenomyosis.Patients:Twenty premenopausal patients with, and20without adenomyosis diagnosed and confirmed by histodiagnosis.Intervention:Immunohistochemistry, Western blot and qPCR were conducted in the eutopic and ectopic endometrium of uteri samples.Main Outcome Measure(s):Location and distribution, protein and mRNA levels of GPR30.Results:GPR30located mostly in the membrane, partly in the cytoplasma of glandular epithelial and stromal cells in patients with and without adenomyosis. GPR30immunoreactivity is higher in the proliferative phase than that in the secretory phase, only in the eutopic endometrium from patients with adenomyosis. Compared with controls, GPR30expressions in the eutopic and ectopic endometrium were significantly higher in protein and mRNA levels in adenomyosis (P<0.001, respectively). There were significantly differences in the protein and mRNA levels of GPR30in the eutopic and ectopic endometrium in adenomyosis (P<0.001respectively).Conclusion:All findings suggest that there was elevated GPR30expression in the eutopic and ectopic endometrium in patients with adenomyosis. Objective:To design and establish lentiviral-vector with shRNA targeting human GPR30and infection conditions in the endometrial stroma cell, and screened effective shRNA against GPR30.Methods:According to RNA interference sequence design principles in the public website, we design four specific targeting human GPR30shRNA sequence and a negative control RNA interference scramble sequence, synthetized by Shanghai JiKai technology co., LTD. And restructure mU6-MCS-Ubi-EGFP, transfect Escherichia coli, positive cloning by PCR and sequencing appraisal, extraction plasmid, package293T cell with lentivirus, measure and calibrate virus titer. In the primary cultured human endometrial stromal cells, according to preliminary experimental principle, to judge infection efficiency by fluorescence microscope, to estabilish the optimal infection condition parameters. Through the PCR and qPCR analysis, detection GPR30mRNA expression level, identify the four shRNA sequence of interference efficiency.Results:Four insert shRNA sequences were correct after extraction plasmid DNA and sequencing, the virus titer were:8×108,5×108,8×108,8×108TU/mL. The infection efficiency was>95%, with the addition of ENi.S. and polybrene, and fifty MOI for human endometrial stromal cells by fluorescence microscope. Through PCR and qPCR analysis after72h, GPR30mRNA level declined significnatly, and four shRNA sequence of interference efficiency were91.53%,81.72%,91.10%and40.65%.Conclusion:1Succesfully design and establish lentiviral-vector with shRNA targeting human GPR30, with higher titer.2Succesfully establish infection conditions in the primary endometrial stroma cell.3Succesfully screen interference efficiency shRNA sequence against GPR30. Objective:To observe the cell proliferation, invasiveness and apoptosis after GPR30RNAi in the primary endometrial stroma cell.Methods:MTT/CCK8, scratch test, and western blot were conducted to detect cell proliferation, invasiveness and apoptosis after GPR30RNAi in the primary endometrial stroma cell.Results:MTT and CCK8showed that the absorbance levels were both significantly lower in GPR30RNAi group than that in non-interference group and negative sequence control group. After GPR30RNAi in the endometrial stroma cell, the scratch was not be repaired in48h, but the scratch appeared to be repared in12h in non-interference group and negative sequence control group, and the scratch showed visible distance after24h, the scratch was completely recover after48h. After GPR30RNAi in the endometrial stroma cell, GPR30and downstream pAkt/Akt protein significantly decreased, there were significantly down-regulation of caspase-3active/pro ratio and bcl-2, and up-regulation of bcl-x, but there was no change in the P53and PARP-1(cleaved p85) expression.Conculsion:1After GPR30RNAi in the endometrial stroma cell, GPR30and downstream pAkt/Akt protein were significantly down-regulation.2After GPR30RNAi in the endometrial stroma cell, cell proliferation was significantly inhibited.3After GPR30RNAi in the endometrial stroma cell, cell invasiveness was significantly inhibited.4After GPR30RNAi in the endometrial stroma cell, there were significantly down-regulation of caspase-3active/pro ratio and bcl-2, and up-regulation of bcl-x.