在长期雌激素耗竭的MCF-7细胞中,截断的WT1蛋白亚型表达增加。

IF 1.6 Q4 ONCOLOGY International Journal of Breast Cancer Pub Date : 2021-11-20 eCollection Date: 2021-01-01 DOI:10.1155/2021/6282514
Saavedra-Alonso Santiago, Zapata-Benavides Pablo, Mendoza-Gamboa Edgar, Chavez-Escamilla Ana Karina, Arellano-Rodríguez Mariela, Rodriguez-Padilla Cristina
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引用次数: 1

摘要

背景:wt1基因编码一种转录因子,该转录因子具有多种具有不同生物学特性的蛋白亚型,能够对参与增殖、分化和凋亡的基因进行正向和负向调节。WT1蛋白在90%以上的乳腺癌中过表达;然而,其在肿瘤进展过程中的作用尚不清楚。方法。在这项工作中,我们分析了WT1亚型在几种具有不同肿瘤标志物状态的乳腺癌细胞中的表达,并使用长期雌激素耗尽培养的MCF-7细胞(MCF-7 ltted细胞)进行了体外实验,最终模拟了从激素依赖性到激素非依赖性的转换过程。此外,还进行了生长动力学、对他莫昔芬的敏感性以及ER和Her2/neu的相对表达分析。结果:western blot初步检测到乳腺癌细胞系ER(+)中有52-54 kDa蛋白异构体的表达,ER(-)中没有WT1蛋白异构体的表达,分析的所有细胞系中均检测到WT1 36-38 kDa蛋白异构体。RT-PCR的选择性剪接分析表明,WT1的17AA (+)/KTS(-)亚型在4个细胞系中最常见。体外实验中,MCF-7细胞雌激素耗竭实验显示,WT1的52-54 kDa亚型在前48小时内表达增加,并持续到第13周,随后,这种表达减少,而WT1的36-38 kDa亚型在前48小时内没有变化,但从第1周开始表达增加,并一直持续到第27周。生长动力学分析显示,与正常条件下培养的MCF-7细胞相比,MCF-7 ltted细胞的增殖能力下降了1.4倍。MCF-7 ltted细胞对他莫昔芬抗增殖作用的敏感性降低(p≤0.05)。通过qRT-PCR分析,收集到第57周的样本显示Her2/neu和ER的相对表达增加。结论:WT1蛋白异构体的调节表现为依赖雌激素受体的WT1蛋白异构体的差异表达。在被归类为晚期细胞的er阴性乳腺癌细胞系中检测到WT1的52-54 kDa缺失和36-38 kDa蛋白亚型的存在。在MCF-7细胞中进行长期雌激素耗尽实验,发现36-38 kDa亚型的表达增加,52-54 kDa亚型的表达减少,这些细胞显示肿瘤标志物ER和Her2/neu的表达增加。与正常条件下的MCF-7细胞相比,MCF-7 ltted细胞增殖低,对他莫昔芬不敏感。这些结果支持了WT1 36-38 kDa亚型与晚期乳腺癌内质网功能缺失之间关系的理论。
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Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background: The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed.

Results: Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER.

Conclusions: Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

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来源期刊
CiteScore
3.40
自引率
0.00%
发文量
25
审稿时长
19 weeks
期刊介绍: International Journal of Breast Cancer is a peer-reviewed, Open Access journal that provides a forum for scientists, clinicians, and health care professionals working in breast cancer research and management. The journal publishes original research articles, review articles, and clinical studies related to molecular pathology, genomics, genetic predisposition, screening and diagnosis, disease markers, drug sensitivity and resistance, as well as novel therapies, with a specific focus on molecular targeted agents and immune therapies.
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