The Effect Of Extracellular Calcium-sensing Receptor On Epithelial Mesenchymal Transitions Of Endometrial Cancer In Hypoxic Microenvironment

Objective:Myometrial invasion and distant metastasis are considered as the most important prognostic factors in endometrial cancer. For this process to occur, epithelial to mesenchymal transition has an important role to play in it. Several studies have shown that hypoxia is deeply related to the epithelial mesenchymal transitions process and calcium-sensing receptor could play a key role in it also. Further studies are needed to elucidate the mechanism of epithelial mesenchymal transitions reducing by hypoxia and illustrated the role of calcium-sensing receptor in it for developing more effective and specific therapies against endometrial cancer progression and metastasis.Methods:1. The expression of HIF-1α, E-cadherin and vimentin in endometrial cancer was detected by immunohistochemistry.2. The change of cytoskeleton and expression of E-cadherin, vimentin after hypoxia or transfection in both endometrial cancer cell line were observed by Immunofluorescence staining.3. The expression of HIF-1 alpha was disturbed by Transient transfection.4. The migration and invasion ability of endometrial cancer after hypoxia or transfection was detected by Transwell model.5. RT-PCR and Western blotting were used to analysis the amplification and expression of HIF-1 a, E-cadherin, Vimentin, Snail and calcium-sensing receptor in Ishikawa cell after hypoxia or transfection.6. After building lentivirus containing silence or overexpression of calcium-sensing receptor gene and transfecting the endometrial cancer cells, fluorescent Fluo-2/AM was used to tracer and monitor the intracellular calcium ions changes with the change of calcium-sensing receptor expression, and RT-PCR, Western blotting and Immunofluorescence staining method were used to detect the expression of epithelial mesenchymal transitions related proteins and functional changes, respectively.7. CCK8 method was used to detect the cell proliferation.Results:1. Compared with normal endometrials, expression of HIF-1α, vimentin increased and expression of E-cadherin decreased in endometrial cancer. The expression of E-cadherin in endometrial cancer was negatively correlated with HIF-1αand vimentin expression.2. Endometrial Cancer cells lose their polarity and adherens junction formation, undergoing a polarity remodeling of the cytoskeleton and eventually obtaining a broadening gap, migratory phenotype with the decreasing expression of E-cadherin and increasing expression of vimentin after hypoxia.3. Amplification and expression of HIF-la, Vimentin and Snail were increased, E-cadherin was decreased with the significantly high expression of calcium-sensing receptor after hypoxia, suggesting endometrial carcinoma acquired epithelial mesenchymal transitions changes and accompanied by the expression of calcium-sensing receptor increase in anoxic environment4. HIF-la enhanced the tumor cell invasion ability and epithelial mesenchymal transitions changes after hypoxia, and blocked by obstructing the expression of HIF-1α via successfully transfected with plasmid containing siRAN-HIF-1α. Meanwhile, HIF-la was negatively regulated by calcium-sensing receptor.5. Intracellular calcium ion flow variation was consistent with the changes of calcium-sensing receptor expression in endometrial cancer cells, respectively. Overexpression calcium-sensing receptor promoted cell apoptosis and suppressed the epithelial to mesenchymal transition formation with increased E-cadherin and decreased vimentin induced by HIF-la, though had no effect on the expression of HIF-la accompanied by the increased expression and changed localization of P-catenin.Conclusion:Epithelial mesenchymal transition was reduced by hypoxia, at least in part dependent on the increasing of HIF-la which negatively regulated by calcium-sensing receptor in endometrial cancer. Hypoxia can also promote the expression of calcium-sensing receptor, and then inhibited the proliferation of endometrial cancer cells and epithelial mesenchymal transitions happened, but its mechanism remains to be further explored for providing a more reliable research foundation to interfere with epithelial mesenchymal transitions of endometrial cancer.