维生素D通过下调谷氨酰胺合成酶抑制乳腺癌细胞增殖  

Vitamin D Inhibits Breast Cancer Cell Proliferation by Down-Regulating Glutamine Synthetase

作　　者：袁惠玲 吴丽华[1] 陈桂林[1] 黄珂铭[1] YUAN Huiling;WU Lihua;CHEN Guilin;HUANG Keming(Department of Mastopathy, Dongguan People’s Hospital Affiliated to Southern Medical University, Dongguan 523000, China)

机构地区：[1]南方医科大学附属东莞人民医院乳腺科,广东东莞523000

出　　处：《标记免疫分析与临床》2022年第1期145-152,共8页Labeled Immunoassays and Clinical Medicine

基　　金：国家自然科学基金项目(编号:81802625);东莞市科技发展重点项目(编号:2018507150011656)。

摘　　要：目的探究维生素D对非三阴性乳腺癌(non-triple negative breast cancer,Non-TNBC)和TNBC癌细胞增殖的影响及分子机制。方法收集TNBC和Non-TNBC患者乳腺组织,原代培养TNBC和Non-TNBC患者乳腺细胞;免疫荧光检测乳腺细胞雌激素受体(ER)、孕激素受体(PR)和HER2蛋白表达;免疫荧光检测Non-TNBC和TNBC乳腺癌组织和乳腺细胞中VDR蛋白表达;CCK-8检测细胞活力变化;流式细胞术检测细胞增殖和细胞周期的变化;光学比色法检测细胞谷氨酰胺合成酶(glutamine synthetase,GS)活力的变化;ELISA检测细胞培养上清和胞内谷氨酰胺的水平;Western blot检测细胞GS和VDR蛋白表达的变化;CHIP-PCR检测VDR对GS的转录调控。结果TNBC患者外周血中维生素D水平和癌组织VDR蛋白表达明显低于Non-TNBC患者(P<0.05)。相比于原代Non-TNBC乳腺癌细胞,原代TNBC患者乳腺癌细胞低表达雌激素受体(ER)、孕激素受体(PR)和HER2蛋白表达;TNBC乳腺癌细胞VDR表达水平明显低于Non-TNBC乳腺癌细胞(P<0.05);VD(10^(-7)~10^(-4)mol/L)可明显剂量依赖性的抑制Non-TNBC乳腺癌细胞和TNBC乳腺癌细胞细胞活力(P<0.05)。相比于0mol/L VD组,10^(3)mol/L的VD组Non-TNBC乳腺癌细胞和TNBC乳腺癌细胞细胞增殖比例明显减少(P<0.05);相比于0mol/L VD组,10^(3)mol/L的VD组Non-TNBC乳腺癌细胞G1/G0期细胞比例明显减少(P<0.05);10^(3)mol/L的VD组TNBC乳腺癌细胞G1/G0期细胞比例明显减少,S期细胞比例明显增加(P<0.05);相比于0mol/L VD组,10^(3)mol/L的VD组Non-TNBC和TNBC乳腺癌细胞谷氨酰胺合成酶活力、谷氨酰胺合成酶蛋白、细胞内和细胞外谷氨酰胺水平表达比例明显减少(P<0.05);相比于0mol/L VD组,10^(3)mol/L的VD组Non-TNBC乳腺癌细胞和TNBC乳腺癌细胞VDR蛋白表达明显增加(P<0.05);10^(3)mol/L的VD组TNBC乳腺癌细胞VDR可转录调控GS基因转录(P<0.05)。结论维生素D通过下调谷氨酰胺合成酶抑制乳腺癌细胞增殖。Objective To explore the effect and molecular mechanism of vitamin D(VD)on the proliferation of non-triple negative breast cancer(Non-TNBC)and TNBC cancer cells.Methods We collected breast tissues from patients with TNBC and Non-TNBC,and cultured primary breast cells from patients with TNBC and Non-TNBC;Estrogen receptor(ER),progesterone receptor(PR)and HER2 protein expressions in breast cells immunofluorescence was detected by immunofluorescence;VDR protein expression in Non-TNBC and TNBC breast cancer tissues and breast cells was detected by immunofluorescence;Cell viability was detected by CCK-8 method;Cell proliferation and cell cycle was detected by flow cytometry;Glutamine synthetase(GS)activity was detected by optical colorimetry method;Intracellular and extracellular glutamine were detected by ELISA;GS and VDR protein expressions were detected by western blot;The transcriptional regulation of GS by VDR was detected by CHIP-PCR.Results VD concentration in peripheral blood and the VDR protein expression in cancer tissues of patients with TNBC were significantly lower than those of patients with Non-TNBC(P<0.05);ER,PR,HER2 and VDR protein expressions in breast cancer cells of primary TNBC breast cancer cells were lower than primary Non-TNBC breast cancer cells(P<0.05);VD(10^(-7)-10^(-4) mol/L)could significantly inhibit the cell viability of Non-TNBC breast cancer cells and TNBC breast cancer cells in a dose-dependent manner(P<0.05).Compared with the 0mol/L VD group,the proliferative proportion of Non-TNBC breast cancer cells and TNBC breast cancer cell in the 10^(3) mol/L VD group was significantly reduced(P<0.05);Compared with the 0mol/L VD group,the proportion of non-TNBC breast cancer cells in the G1/G0 phase of the 10^(3) mol/L VD group was significantly reduced(P<0.05);The proportion of TNBC breast cancer cells in the G1/G0 phase of the 10^(3) mol/L VD group was significantly reduced,and the proportion of cells in the S phase was significantly increased(P<0.05);Compared with 0mol/L VD group,Non-TNBC an

关 键 词：维生素D 三阴性乳腺癌 维生素受体 谷氨酰胺合成酶 细胞增殖 

分 类 号：Q565[生物学—生物化学] R737.9[医药卫生—肿瘤]