甲基化介导的 miR-125a-5p 沉默通过诱导自噬促进乳腺癌进展。

Methylation-mediated silencing of miR-125a-5p facilitates breast cancer progression by inducing autophagy.

机构信息

Department of Clinical Biochemistry, School of Medicine, Cellular and Molecular Research Center, Medical Basic Science Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Mol Biol Rep. 2022 Jul;49(7):6325-6339. doi: 10.1007/s11033-022-07440-y. Epub 2022 Apr 29.

DOI:10.1007/s11033-022-07440-y

PMID:35486286

Abstract

BACKGROUND

microRNA-125a-5p (miR-125a) is a tumor suppressor gene whose role in autophagy remains poorly understood. In the current study, we aimed to investigate the methylation status of miR-125a, its transfection into SK-BR3 cells, and its effects on autophagy.

METHODS

Sixty samples of tumor and non-tumor adjacent tissue were collected and the methylation status of miR-125a was evaluated by methylation-specific PCR (MSP). The effect of 5-Aza-dC on miR-125a expression was investigated in the SK-BR3 cells. Cells were also transfected with miR-125a mimic/antimiR. The expression of miR-125a and its target genes was evaluated by Real-Time PCR. Protein levels of ATG5 and LC3 were assessed by Western blotting. HER2 expression was investigated by immunocytochemistry (ICC).

RESULTS

The data showed that the miR-125a promoter CpG Island was significantly hypermethylated in breast cancer tissues (p < 0.01) and in SK-BR3 cells. The 5-Aza-dC could significantly increase miR-125a expression by decreasing its methylation (p < 0.05). In addition, Western blot analysis indicated the expression of ATG5 and LC3 II/ LC3I, as autophagy biomarkers, was significantly reduced in SK-BR3 cells transfected with miR-125a (p < 0.05).

CONCLUSIONS

Our data showed miR-125a expression was significantly decreased in tumor tissues due to its promoter hypermethylation. Overexpression of miR-125a was associated with a reduction in autophagy, which could provide a new therapeutic avenue for advanced-stage breast cancer treatment.

摘要

背景

microRNA-125a-5p（miR-125a）是一种肿瘤抑制基因，其在自噬中的作用仍知之甚少。在本研究中，我们旨在研究 miR-125a 的甲基化状态、转染 SK-BR3 细胞及其对自噬的影响。

方法

收集 60 例肿瘤和非肿瘤相邻组织样本，通过甲基化特异性 PCR（MSP）评估 miR-125a 的甲基化状态。研究 5-Aza-dC 对 SK-BR3 细胞中 miR-125a 表达的影响。还转染了 miR-125a 模拟物/反义 miR。通过实时 PCR 评估 miR-125a 及其靶基因的表达。通过 Western blot 评估 ATG5 和 LC3 的蛋白水平。通过免疫细胞化学（ICC）研究 HER2 的表达。

结果

数据显示，乳腺癌组织和 SK-BR3 细胞的 miR-125a 启动子 CpG 岛明显过度甲基化（p<0.01）。5-Aza-dC 可通过降低其甲基化来显著增加 miR-125a 的表达（p<0.05）。此外，Western blot 分析表明，转染 miR-125a 的 SK-BR3 细胞中自噬标志物 ATG5 和 LC3 II/LC3I 的表达明显降低（p<0.05）。