P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time
P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time
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Pâ288 低剂量二甲双胍治疗后人子宫内膜癌细胞系基因和蛋白质表达随时间的变化
DOI：
10.1093/humrep/deab130.287
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发表时间：
2021
影响因子：
6.1
通讯作者：
Germeyer
中科院分区：
文献类型：
--
作者：
Thüner;Jauckus;Strowitzki;Germeyer
二甲双胍治疗是否会导致子宫内膜癌细胞系中特定基因和蛋白质的时间表达变化？概要答案研究了三种不同基因和蛋白的表达，它们都显示出随时间的变化。已知子宫内膜癌（EC）是绝经后妇女最常见的恶性肿瘤之一。雌激素对子宫内膜的长期影响常见于肥胖或多囊卵巢综合征（PCOS）以及II型糖尿病（T2 DM）女性，是EC发生和进展的众所周知的风险因素。二甲双胍是一种双胍类药物，用于治疗T2 DM患者和PCOS女性超说明书使用。此外，二甲双胍在包括EC在内的各种癌症类型中显示出抗肿瘤和抗增殖作用。在这方面，BCL 2L 11、CDH 1和CDKN 1A在凋亡途径、增殖和侵袭过程中起重要作用。研究设计、大小、持续时间EC细胞在正常（5.5 mmol/L）或高（17 mmol/L）葡萄糖培养基中培养，补充10 nmol/L β-雌二醇。用低剂量二甲双胍（1.0 mmol/L）处理细胞2、6、24、48和168 h（7 d）。此外，EC细胞用二甲双胍和胰岛素（100 ng/mL）的组合处理或保持未处理。五个独立的实验完成和未处理的细胞作为controls.Participants/材料，设置，方法该研究是使用两种不同的人EC细胞系完成的。HEC-1A代表雌激素非依赖性EC，而石川代表更常见的雌激素依赖性EC。提取蛋白质，用BCA测定定量，并通过蛋白质印迹分析BCL 2L 11、CDH 1和CDKN 1A的蛋白质表达。此外，提取总RNA，转录成cDNA，并进行Taqman实时PCR以测量相关基因的表达，使用倍数变化（FC）作为参数。主要结果和机会的作用通过RT-PCR分析，所选基因的表达在两种细胞系中随时间的变化如下：6 h后，二甲双胍诱导BCL 2L 11（FC = 0.7）和CDH 1（FC = 0.75）表达降低，而CDKN 1A表达略有增加（FC = 0.95-1.35）。24 h后，正常葡萄糖组BCL 2L 11表达增加（FC = 1.3，高糖：FC = 0.93），二甲双胍和高糖联合组CDH 1表达降低（FC = 0.7，正常葡萄糖：FC = 1.1）。24小时后，二甲双胍使两种细胞系中的CDKN 1A表达增加（FC = 1.2-1.8）。二甲双胍处理48小时后，所有三种基因的表达仅发生轻微变化（FC = 0.9-1.0）。7天后，观察到高糖和二甲双胍的组合（即，类似于肥胖T2 DM患者）在存在和不存在胰岛素的情况下导致BCL 2L 11、CDH 1和CDKN 1A的表达增加（FC = 1.4-2.9），而二甲双胍在正常葡萄糖培养基中诱导CDH 1和CDKN 1A的表达减少（FC = 0.5-0.75）。BCL 2L 11、CDH 1和CDKN 1A的表达也在蛋白水平上进行了研究。局限性、注意事项由于该研究是在体外进行的，因此该结果不能直接应用于二甲双胍治疗患者。此外，包括更多时间点的进一步研究将更精确地表明基因和蛋白质表达随时间的变化。研究结果的更广泛意义：这是第一个体外研究显示BCL 2L 11，CDH 1和CDKN 1A表达的时间变化，这些基因与低剂量二甲双胍引起的肿瘤发生有关。
Study questionDoes metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines?Summary answerThe expression of three different genes and proteins was investigated, of which all displayed changes over time.What is known alreadyEndometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes.Study design, size, durationThe EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls.Participants/materials, setting, methodsThe study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter.Main results and the role of chanceThe expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well.Limitations, reasons for cautionThe results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time.Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin …