G蛋白偶联受体30对子宫内膜癌细胞增殖及凋亡的影响及其机制探讨

Effects of G protein-coupled receptor 30 on proliferation and apoptosis of endometrial carcinoma cells and its possible mechanism

目的:研究G蛋白偶联受体30(G protein-coupled receptor 30,GPR30)对子宫内膜癌细胞增殖及凋亡能力的影响,并探讨其可能的作用机制。方法:将靶向GPR30的shRNA(shRNA-GPR30)或携带有GPR30全基因的慢病毒(LV-GPR30)分别导入子宫内膜癌Ishikawa和KLE细胞中,构建沉默GPR30和过表达GPR30的细胞株,然后用蛋白质印迹法检测转染效率。采用CCK-8法检测沉默或过表达GPR30对子宫内膜癌细胞增殖能力的影响,采用FCM法检测沉默或过表达GPR30对子宫内膜癌细胞凋亡能力的影响。采用实时荧光定量PCR及蛋白质印迹法检测沉默或过表达GPR30对内质网应激标志蛋白葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)和内质网应激的未折叠蛋白反应信号通路关键分子蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)、肌醇需求酶1(inositol-requiring enzyme 1,IRE1)、活化转录因子6(activating transcription factor 6,ATF6)的表达水平变化。结果:GPR30分别在Ishikawa和KLE细胞中被沉默或过表达。沉默GPR30后,子宫内膜癌细胞的增殖能力下降,凋亡率升高(P值均<0.01);过表达GPR30后,子宫内膜癌细胞的增殖能力增强,凋亡率降低(P值均<0.01)。沉默GPR30可下调子宫内膜癌细胞中GRP78、PERK、IRE1和ATF6的表达水平,过表达GPR30可上调子宫内膜癌中GRP78、PERK、IRE1和ATF6的表达(P值均<0.01)。结论:沉默GPR30可抑制子宫内膜癌细胞的增殖能力,促进细胞凋亡;过表达GPR30可促进子宫内膜癌细胞的增殖能力,并抑制细胞凋亡。推测该机制可能与内质网应激途径的未折叠蛋白反应有关。

Objective:To investigate the effects of G protein-coupled receptor 30(GPR30)on the proliferation and apoptosis of human endometrial cancer cells,and to explore its possible mechanism.Methods:ShRNA targeted GPR30(shRNA-GPR30)and lentivirus carrying GPR30(LV-GPR30)were used to transfected or infected into endometrial cancer cells,respectively.GPR30 silenced and overexpressed cells were constructed to detect the transfection efficiency by Western blotting.The effect of silencing or overexpression GPR30 on the proliferation of endometrial carcinoma cells was detected by CCK-8 method.The effects of GPR30 silence or overexpression on the apoptosis of endometrial cancer cells were detected by flow cytometry.The expressions of endoplasmic reticulum stress marker glucose-regulated protein-78(GRP78)and the key molecules including protein kinase R-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme 1(IRE1),and activating transcription factor 6(ATF6)in unfolded protein reactive signaling pathway in the two endometrial cancer cells with GPR30 silence or overexpression were detected by real-time fluorescence quantitative PCR and Western blotting.Results:GPR30 was silenced and overexpressed in Ishikawa and KLE cells transfected with shRNA-GPR30 and infected with LV-GPR30,respectively.After GPR30 was silenced,the proliferative capacity of endometrial cancer cells was decreased and the apoptosis was increased(all P<0.01).On the contrary,after GPR30 was overexpressed,the proliferative capacity of endometrial cancer cells was increased and the apoptosis was decreased(all P<0.01).GPR30 silencing could down-regulate the expressions of GRP78,PERK,IRE1,and ATF6 in endometrial cancer cells(all P<0.01),while the overexpressing GPR30 could up-regulate the expressions of GRP78,PERK,IRE1,and ATF6 in endometrial cancer cells(all P<0.01).Conclusion:Silencing GPR30 can inhibit the proliferation of endometrial cancer cells and promote apoptosis.Overexpression of GPR30 can promote the proliferation of endometrial cancer cells and inhibit apoptosis.The mechanism may be related to the unfolded protein response in endoplasmic reticulum stress pathway.