CAJ | 학술논문

目的探讨二甲双胍对雌激素诱导的子宫内膜癌细胞增殖及ER表达的影响.方法不同浓度(分别为0.5、1、5、10、15、20、25、30 mmol/L)二甲双胍作用于子宫内膜癌细胞系Ishikawa和HEC-1A细胞不同时间(分别为24、48、72 h)后,采用四甲基偶氮唑蓝(MTT)比色法检测Ishikawa和HEC-1A细胞的增殖情况.17β雌二醇(1×10-6mol/L)单独及联合二甲双胍(5 mmol/L)分别作用Ishikawa和HEC-1A细胞24 h后,通过5-溴脱氧尿嘧啶核苷(BrdU)标记法检测Ishikawa和HEC-1A细胞的增殖情况;不同浓度(分别为1、5、15 mmol/L)二甲双胍作用24 h后,实时荧光定量PCR技术检测Ishikawa和HEC-1A细胞中原癌基因c-fos和c-myc mRNA的表达水平,蛋白印迹法检测Ishikawa和HEC-1A细胞中ERα和ERβ蛋白的表达水平.结果 MTT比色法检测显示,与对照(仅加入培养基)细胞比较,上述不同浓度的二甲双胍作用不同时间后,Ishikawa和HEC-1A细胞增殖的抑制率均明显增加(P＜0.05),且随着二甲双胍浓度的增加及作用时间的延长,其抑制作用呈明显的剂量及时间依赖性(P＜0.05).BrdU标记法检测显示,17β雌二醇联合二甲双胍作用后Ishikawa、HEC-1A细胞的增殖率分别为(62±7)％、(72±6)％,分别与17β雌二醇单独作用后的Ishikawa、HEC-1A细胞的增殖率[分别为(124±16)％、(109±5)％]比较,差异均有统计学意义(P＜0.01).实时荧光定量PCR技术检测显示,不同浓度(分别为1、5、15 mmol/L)二甲双胍作用后,Ishikawa、HEC-1A细胞中c-fos和c-myc mRNA表达水平均逐渐降低,除HEC-1A细胞中1 mmol/L的二甲双胍作用后c-myc mRNA表达水平与相应对照(即二甲双胍浓度为0)细胞比较差异无统计学意义(P=0.074)外,其余浓度分别与相应对照比较差异均有统计学意义(P＜0.05);蛋白印迹法检测显示,随着二甲双胍浓度的增加,Ishikawa、HEC-1A细胞中ERα蛋白的表达水平逐渐下降,而ERβ蛋白的表达水平逐渐增加,其浓度为5 mmol/L和15 mmol/L时分别与相应对照细胞比较,差异均有统计学意义(P＜0.05).结论二甲双胍能抑制雌激素对子宫内膜癌细胞的促增殖作用,其作用机制可能与二甲双胍调节ER的表达进而影响原癌基因表达有关. 目的探討二甲雙胍對雌激素誘導的子宮內膜癌細胞增殖及ER錶達的影響.方法不同濃度(分彆為0.5、1、5、10、15、20、25、30 mmol/L)二甲雙胍作用于子宮內膜癌細胞繫Ishikawa和HEC-1A細胞不同時間(分彆為24、48、72 h)後,採用四甲基偶氮唑藍(MTT)比色法檢測Ishikawa和HEC-1A細胞的增殖情況.17β雌二醇(1×10-6mol/L)單獨及聯閤二甲雙胍(5 mmol/L)分彆作用Ishikawa和HEC-1A細胞24 h後,通過5-溴脫氧尿嘧啶覈苷(BrdU)標記法檢測Ishikawa和HEC-1A細胞的增殖情況;不同濃度(分彆為1、5、15 mmol/L)二甲雙胍作用24 h後,實時熒光定量PCR技術檢測Ishikawa和HEC-1A細胞中原癌基因c-fos和c-myc mRNA的錶達水平,蛋白印跡法檢測Ishikawa和HEC-1A細胞中ERα和ERβ蛋白的錶達水平.結果 MTT比色法檢測顯示,與對照(僅加入培養基)細胞比較,上述不同濃度的二甲雙胍作用不同時間後,Ishikawa和HEC-1A細胞增殖的抑製率均明顯增加(P＜0.05),且隨著二甲雙胍濃度的增加及作用時間的延長,其抑製作用呈明顯的劑量及時間依賴性(P＜0.05).BrdU標記法檢測顯示,17β雌二醇聯閤二甲雙胍作用後Ishikawa、HEC-1A細胞的增殖率分彆為(62±7)％、(72±6)％,分彆與17β雌二醇單獨作用後的Ishikawa、HEC-1A細胞的增殖率[分彆為(124±16)％、(109±5)％]比較,差異均有統計學意義(P＜0.01).實時熒光定量PCR技術檢測顯示,不同濃度(分彆為1、5、15 mmol/L)二甲雙胍作用後,Ishikawa、HEC-1A細胞中c-fos和c-myc mRNA錶達水平均逐漸降低,除HEC-1A細胞中1 mmol/L的二甲雙胍作用後c-myc mRNA錶達水平與相應對照(即二甲雙胍濃度為0)細胞比較差異無統計學意義(P=0.074)外,其餘濃度分彆與相應對照比較差異均有統計學意義(P＜0.05);蛋白印跡法檢測顯示,隨著二甲雙胍濃度的增加,Ishikawa、HEC-1A細胞中ERα蛋白的錶達水平逐漸下降,而ERβ蛋白的錶達水平逐漸增加,其濃度為5 mmol/L和15 mmol/L時分彆與相應對照細胞比較,差異均有統計學意義(P＜0.05).結論二甲雙胍能抑製雌激素對子宮內膜癌細胞的促增殖作用,其作用機製可能與二甲雙胍調節ER的錶達進而影響原癌基因錶達有關.
목적 탐토이갑쌍고대자격소유도적자궁내막암세포증식급ER표체적영향.방법 불동농도(분별위0.5、1、5、10、15、20、25、30 mmol/L)이갑쌍고작용우자궁내막암세포계Ishikawa화HEC-1A세포불동시간(분별위24、48、72 h)후,채용사갑기우담서람(MTT)비색법검측Ishikawa화HEC-1A세포적증식정황.17β자이순(1×10-6mol/L)단독급연합이갑쌍고(5 mmol/L)분별작용Ishikawa화HEC-1A세포24 h후,통과5-추탈양뇨밀정핵감(BrdU)표기법검측Ishikawa화HEC-1A세포적증식정황;불동농도(분별위1、5、15 mmol/L)이갑쌍고작용24 h후,실시형광정량PCR기술검측Ishikawa화HEC-1A세포중원암기인c-fos화c-myc mRNA적표체수평,단백인적법검측Ishikawa화HEC-1A세포중ERα화ERβ단백적표체수평.결과 MTT비색법검측현시,여대조(부가입배양기)세포비교,상술불동농도적이갑쌍고작용불동시간후,Ishikawa화HEC-1A세포증식적억제솔균명현증가(P＜0.05),차수착이갑쌍고농도적증가급작용시간적연장,기억제작용정명현적제량급시간의뢰성(P＜0.05).BrdU표기법검측현시,17β자이순연합이갑쌍고작용후Ishikawa、HEC-1A세포적증식솔분별위(62±7)％、(72±6)％,분별여17β자이순단독작용후적Ishikawa、HEC-1A세포적증식솔[분별위(124±16)％、(109±5)％]비교,차이균유통계학의의(P＜0.01).실시형광정량PCR기술검측현시,불동농도(분별위1、5、15 mmol/L)이갑쌍고작용후,Ishikawa、HEC-1A세포중c-fos화c-myc mRNA표체수평균축점강저,제HEC-1A세포중1 mmol/L적이갑쌍고작용후c-myc mRNA표체수평여상응대조(즉이갑쌍고농도위0)세포비교차이무통계학의의(P=0.074)외,기여농도분별여상응대조비교차이균유통계학의의(P＜0.05);단백인적법검측현시,수착이갑쌍고농도적증가,Ishikawa、HEC-1A세포중ERα단백적표체수평축점하강,이ERβ단백적표체수평축점증가,기농도위5 mmol/L화15 mmol/L시분별여상응대조세포비교,차이균유통계학의의(P＜0.05).결론 이갑쌍고능억제자격소대자궁내막암세포적촉증식작용,기작용궤제가능여이갑쌍고조절ER적표체진이영향원암기인표체유관.
Objective To assess the effects of metformin on estrogen-induced proliferation of human endometrial cancer cell lines and investigate whether metformin could regulate the expression of ER and estrogen-dependent proliferative genes.Methods Human endometrial cancer cell lines Ishikawa and HEC-1A underwent treatment with metformin at various concentrations (0.5,1,5,10,15,20,25 and 30 mmol/L) for different durations (24,48 and 72 hours),followed by assessment of cell proliferation by methyl thiazolyl tetrazolium (MTT) assay.Ishikawa and HEC-1A cells were exposed to 17β-estradiol (1× 10-6mol/L) alone or in combination with metformin (5 mmol/L) for 24 hours.Cell proliferation was assessed by 5-bromo-2-deoxyuridine (BrdU) assay.Twenty-four hours after metformin treatment at the concentrations of 1,5 and 15 mmol/L,the expression levels of estrogen-dependent proliferative genes c-fos and c-myc were determined by real-time quantitative fluorescent PCR (real-time FQ-PCR).Western blot analysis was performed to assess the effects of metformin on the expressions of estrogen receptors.Results As revealed by MTT assay,at different time points of metformin treatment at different concentrations,the proliferation rates of both cell lines were inhibited in a dose-dependent and time-dependent manner between metformin groups and the control group (P＜0.05).BrdU assay showed that the proliferation rate of Ishikawa and HEC-1A cells exposed to 17β-estradiol (1 × 10-6 mol/L) in combination with metformin (5 mmol/L) was (62±7)％ and (72±6)％,respectively,while that in 17[β-estradiol groups was (124± 16)％ and (109±5)％,respectively,with significantly statistical differences (P＜0.01).By real-time FQ-PCR tested,the expression levels of c-fos and c-myc in both cell lines gradually declined subsequent to metformin treatment at different concentrations (1,5 and 15 mmol/L).As compared with the control group,the c-myc and c-fos expressions in both cell lines in metformin groups had significant differences (P＜0.05) except for the c-myc expression of the concentration of 1 mmol/L in HEC-1A cell line (P=0.074).Western blot analyses showed that with the increasing concentrations of metformin,the ERα expression was markedly down-regulated,while ERβ expression was up-regulated in the metformin group at the concentrations of 5 mmol/L and 15 mmol/L,compared to those at the control group,there were significant differences between them,respectively (all P＜0.01).Conclusion Metformin could inhibit estrogen-mediated proliferation of human endometrial cancer cells,which might be correlated with its regulation of the expressions of estrogen receptors and estrogen-dependent proliferative genes.