二甲双胍对子宫肌瘤组织中雌激素合成相关酶及ER表达的调控作用

摘要

目的 探讨二甲双胍对子宫肌瘤组织中雌激素合成相关酶和ER表达的调控作用.方法(1)收集2016年12月—2017年1月在北京大学第一医院妇产科因子宫肌瘤行子宫全切除术且经术后病理检查确诊为子宫肌瘤的患者17例,留取其子宫肌瘤组织及周围正常子宫肌层组织(距离肌瘤组织2 cm以上),组成自身配对,采用逆转录(RT)-PCR技术检测子宫肌瘤和正常子宫肌层组织中雌激素合成相关酶[包括细胞色素P450胆固醇侧链裂解酶(P450scc)、细胞色素P450 17α-羟化酶(P450c17)、3β-羟基类固醇脱氢酶2(3β-HSD-2)、17β-羟基类固醇脱氢酶1(17β-HSD-1)和芳香酶(P450arom)]及ER(包括ERα和ERβ)mRNA的表达.(2)原代子宫肌瘤细胞培养成功并鉴定后,取第3代子宫肌瘤细胞,实验组加入不同浓度(分别为10、50、100 μmol/L)的二甲双胍,对照组加入去离子水,采用RT-PCR技术检测两组子宫肌瘤细胞中3β-HSD-2和P450arom及ERα和ERβ mRNA的表达.结果(1)子宫肌瘤组织中P450scc、P450c17、3β-HSD-2、17β-HSD-1、P450arom mRNA的中位表达水平分别为112、4、13、42、194,正常子宫肌层组织分别为114、5、11、32、6,与正常子宫肌层组织比较,子宫肌瘤组织中3β-HSD-2和P450arom mRNA的表达水平明显升高(P<0.05),而P450scc、P450c17、17β-HSD-1 mRNA的表达水平无明显变化(P>0.05).子宫肌瘤组织中ERα、ERβ mRNA的中位表达水平分别为208、116,正常子宫肌层组织分别为24、95,与正常子宫肌层组织比较,子宫肌瘤组织中ERα mRNA的表达水平明显升高(P=0.001),而ERβ mRNA的表达水平无明显变化(P=0.193).(2)实验组分别经浓度为10、50和100 μmol/L的二甲双胍处理后,子宫肌瘤细胞中P450arom mRNA的表达水平分别为9±4、8±5和8±3,分别与对照组(16±5)比较,差异均有统计学意义(P<0.05);实验组中不同浓度的二甲双胍处理后,子宫肌瘤细胞中3β-HSD-2 mRNA的表达水平分别与对照组比较均无明显变化(P>0.05).实验组不同浓度二甲双胍处理后,子宫肌瘤细胞中ERα和ERβ mRNA的表达水平分别与对照组比较,差异均无统计学意义(P>0.05).结论 二甲双胍可在转录水平下调子宫肌瘤细胞中P450arom的表达,从而通过减少雌激素的合成抑制子宫肌瘤的生长.%Objective To elucidate whether metformin could regulate the mRNA expression level of estrogen synthetase and ER in human uterine leiomyoma tissues. Methods (1) Seventeen pairs of uterine leiomyoma tissues and adjacent myometrium (>2 cm) were collected from patients underwent hysterectomy in Peking University First Hospital between December 2016 and January 2017. Real-time PCR was used to measure the mRNA expression level of estrogen synthetase [including cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), cytochrome P450 17 α-hydroxylase (P450c17), 3-beta-hydroxysteroid dehydrogenase type 2 (3β-HSD-2), 17-beta-hydroxysteroid dehydrogenase type 1 (17β-HSD-1) and aromatase cytochrome P450 (P450arom)] and ER (including ERα and ERβ) in the uterine leiomyoma tissues and adjacent myometrium. (2) Uterine leiomyoma cells derived from uterine leiomyoma tissues were identified by immunocytochemistry method and cultured to the third generation. The treatment groups were cultured with different concentrations of metformin (10, 50 and 100 μmol/L) for 48 hours, and the control group was cultured with deionized water for 48 hours. The mRNA expression level of estrogen synthetase and estrogen receptor subtypes were measured by real-time PCR. Results (1) P450scc, P450c17, 3β-HSD-2, 17β-HSD-1, P450arom mRNA median expression levels were 112, 4, 13, 42 and 194 in the uterine leiomyoma tissues, and were respectively 114, 5, 11, 32 and 6 in the myometrium. Compared to those of the myometrium, 3β-HSD-2 and P450arom mRNA expression levels in the uterine leiomyoma tissue were significantly higher (P<0.05), while there were no significant change of mRNA expression levels among P450scc, P450c17 and 17β-HSD-1 (P>0.05). ERα and ERβ mRNA median expression levels were 208 and 116 in the uterine leiomyoma tissues, and were 24 and 95 in the myometrium. Compared to that of the myometrium, ERα mRNA level in the uterine leiomyoma tissue was significantly higher (P=0.001), while there were no significant change of ERβ mRNA level (P=0.193). (2) After cultured with different concentrations of metformin (10, 50 and 100 μmol/L), the P450arom mRNA levels in the uterine leiomyoma tissues were 9 ± 4, 8 ± 5 and 8 ± 3 respectively in the treatment groups and was 16 ± 5 in the control group. Compared to that of the control group, P450arom mRNA expression levels in the treatment groups were significantly declined (P<0.05). There were no significant different change of mRNA expression levels among 3β-HSD-2, ERα and ERβ between the treatment groups and the control group (P>0.05). Conclusions Metformin could down-regulate the mRNA expression level of aromatase in the uterine leiomyoma cells. These results indicate that metformin may inhibit the local estrogen synthesis and therefore suppress the development of uterine leiomyoma.