GPR30与AMF正向反馈调控促进子宫内膜癌进展的机制研究

Positive feedback regulation of GPR30 and AMF promotes the progression of endometrial cancer

目的:探讨子宫内膜癌(EC)中G蛋白偶联跨膜受体30(GPR30)与自分泌移动因子(AMF)正向反馈的调控机制。方法:免疫组化和免疫荧光法检测EC患者子宫内膜组织中AMF和GPR30的表达情况。Western blot法检测雌激素及GPR30特异激动剂G1对RL95-2细胞、KLE细胞及SPEC-2细胞AMF分泌的调控。使用shRNA干扰、qRT-PCR、Western blot及细胞免疫荧光验证雌激素膜受体GPR30介导的AMF表达和分泌。qRT-PCR和Western blot法检测AMF刺激前后细胞GPR30的表达情况。使用信号通路蛋白芯片筛选AMF表达下调前后G蛋白相关信号通路变化,并通过Western blot验证。结果:AMF和GPR30在内膜癌组织中呈高表达,且两者表达水平呈正相关。雌激素刺激后,子宫内膜癌细胞分泌的AMF均显著增多,且与雌激素浓度呈正相关。GPR30特异激动剂G1上调了AMF的分泌。干扰GPR30表达致AMF的表达和分泌下降。外源性AMF刺激可上调GPR30 mRNA及蛋白水平。AMF表达下调后MAPK-ERK信号通路明显受到抑制,同时阻断MAPK-ERK信号通路可消减AMF对GPR30的上调作用。结论:内膜癌细胞中雌激素通过GPR30能显著上调AMF的表达及分泌; AMF可激活MAPK信号通路正向反馈调控GPR30表达。E2-GPR30-AMF轴为内膜癌雌激素致瘤理论提供重要补充,进而为寻找能阻断雌激素作用靶点关键因子的分子靶向治疗提供可靠的实验依据。

Objective:To investigate the mechanism of positive feedback of GPR30 and AMF in endometrial cancer.Methods:The expressions of AMF and GPR30 in endometrial tissues of EC patients were detected by immunohistochemistry and immunofluorescence.In RL95-2 cells,KLE cells and SPEC-2 cells,Western blot was used to examine the secretion of AMF stimulated by estrogen and GPR30-specific agonist G1.We used shRNA interference,qRT-PCR,Western blot,and cellular immunofluorescence to test the estrogen receptor GPR30-mediated AMF expression and secretion.Real-time quantitative PCR and Western blot were used to detect the expression of GPR30 after AMF stimulation.The signal pathway protein microarray was used to screen changes of G-protein related signal pathway after AMF knock-down,and Western blot was used to verify the changes.Result:AMF and GPR30 were highly expressed in endometrial cancer,and their expression levels were positively correlated between AMF and GPR30.After estrogen stimulation,AMF secreted by endometrial cancer cells were significantly increased and positively correlated with estrogen concentrations.Apart from this,GPR30 specific agonist G1 up-regulated AMF secretion.Interfering with GPR30 expression caused a decrease in the expression and secretion of AMF.Exogenous AMF stimulation upregulates GPR30 mRNA and protein levels.Decreased expression of AMF causes significantly inhibition of MAPK-ERK signaling pathway,while blocking MAPK-ERK can reduce the upregulation of GPR30 by AMF.Conclusions:In endometrial cancer cells,estrogen can significantly up-regulate the expression and secretion of AMF through GPR30,and AMF can activate the MAPK signaling pathway to positively regulate the expression of GPR30.The E 2-GPR30-AMF axis provides an important complement to the estrogen oncogenic theory of endometrial cancer,and provides a reliable experimental basis for the search for molecular targeted therapy of endometrial cancer.