背景:乳腺癌(BC)是女性中最常见的癌症。雌激素阳性(ER+)和三阴性BC细胞(TNBC)的治疗方法不同,随后可能会影响癌症生物标志物。如H19和端粒酶,是不列颠哥伦比亚省研究中散发出来的喜悦。例如,全反式维甲酸(ATRA)可以代表这些癌基因的有效调节因子,调节microRNAs,主要是let-7a微小RNA(miR-let-7a),靶向糖酵解途径,主要是丙酮酸激酶M2(PKM2)和乳酸脱氢酶A(LDHA)。这里,我们调查了ATRA在H19,端粒酶,miR-let-7a,和糖酵解酶在ER+和TNBC细胞中的调节。
方法:用5µMATRA和/或100nM氟维司群处理MCF-7和MDA-MB-231细胞。然后,用H19或hTERTsiRNA转染ATRA处理的或对照的MCF-7细胞。之后,用雌激素受体αER(α)或βER(β)表达质粒转染ATRA处理或未处理的MDA-MB-231细胞。通过RT-qPCR评估RNA表达,和蛋白质通过蛋白质印迹进行评估。使用NADH/LDH偶联酶测定法测量PKM2活性,端粒酶活性通过定量端粒重复扩增方案测定进行评估。使用学生t检验或单向ANOVA分析来自重复的数据。
结果:我们的结果表明,MCF-7细胞比MDA-MB-231细胞对ATRA的反应更快。在MCF-7细胞中,ATRA和/或氟维司群降低ER(α),H19,端粒酶,PKM2和LDHA,而ER(β)和miR-let-7a增加。有或没有ATRA治疗的H19或hTERT敲低显示与ATRA治疗后获得的结果相似,发现了H19和hTERT之间的潜在互连。然而,在MDA-MB-231细胞中,在ATRA和/或氟维司群之后调节上述基因的RNA表达,对蛋白质和活性水平没有显著影响。MDA-MB-231细胞中ER(α)或ER(β)过表达诱导端粒酶活性,PKM2和LDHA表达,其中ATRA治疗结合质粒转染降低了糖酵解酶的表达。
结论:据我们所知,我们的研究首次通过miR-let-7a阐明ER+BC细胞中雌激素受体和糖酵解酶之间新的潜在相互作用.
BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in
H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.
METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either
H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student\'s t-test or one-way ANOVA was used to analyze data from replicates.
RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α),
H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased.
H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between
H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.
CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.