Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran.
J Cell Biochem. 2021 Apr;122(3-4):385-393. doi: 10.1002/jcb.29867. Epub 2020 Nov 8.
Epithelial-mesenchymal transition (EMT) is a biological event, which critically regulates migration and invasion of cancer cells. EMT is regulated by several protein and nonprotein factors (such as noncoding RNAs). HOTAIR is an oncogenic long noncoding RNA that stimulates EMT in cancers. In the current study, we investigated the effect of metformin on EMT behavior and HOTAIR expression in MDA-MB-231 breast cancer cells. The minimal effective concentrations of metformin (10 and 20 mM) were obtained by the MTT test. Cell migration and invasion in the metformin-containing medium were assayed in the scratch assay and transwell test. Meaningful decreases in both cell migration and invasion were observed in the presence of metformin. Vimentin, snail, β-catenin, and HOTAIR transcripts were quantified by real-time polymerase chain reaction (PCR). Reduction in the expression of vimentin, β-catenin, and HOTAIR was detected as the result of metformin treatment, but the snail showed a constant expression. Western blottingrevealed the downregulation of vimentin and β-catenin proteins. HOTAIR promoter methylation pattern was also investigated in metformin-exposed cells using bisulfite sequencing PCR which the result showed differences in the methylation profile of CpG islands between the treated and untreated cells. In conclusion, metformin modulated oncogenic expression of the HOTAIR gene in the MDA-MB-231 cells. This downregulation was associated with the modification of promoter methylation patterns. Since HOTAIR induces EMT in breast cancer, HOTAIR decline might be one of the mechanisms by which metformin reverses EMT.
上皮-间质转化 (EMT) 是一种生物学事件，它对癌细胞的迁移和侵袭具有关键调节作用。EMT 受多种蛋白和非蛋白因素（如非编码 RNA）的调节。HOTAIR 是一种致癌的长非编码 RNA，可刺激癌症中的 EMT。在本研究中，我们研究了二甲双胍对 MDA-MB-231 乳腺癌细胞 EMT 行为和 HOTAIR 表达的影响。通过 MTT 试验获得二甲双胍的最小有效浓度（10 和 20mM）。在划痕试验和 Transwell 试验中检测二甲双胍培养基中细胞的迁移和侵袭。在二甲双胍存在的情况下，观察到细胞迁移和侵袭均明显减少。通过实时聚合酶链反应 (PCR) 定量检测波形蛋白、snail、β-catenin 和 HOTAIR 转录本。作为二甲双胍处理的结果，检测到波形蛋白、β-catenin 和 HOTAIR 的表达减少，但 snail 表达恒定。Western blot 显示波形蛋白和 β-catenin 蛋白下调。还使用亚硫酸氢盐测序 PCR 研究了二甲双胍暴露细胞中的 HOTAIR 启动子甲基化模式，结果表明处理和未处理细胞的 CpG 岛甲基化模式存在差异。总之，二甲双胍调节了 MDA-MB-231 细胞中 HOTAIR 基因的致癌表达。这种下调与启动子甲基化模式的修饰有关。由于 HOTAIR 在乳腺癌中诱导 EMT，HOTAIR 的下降可能是二甲双胍逆转 EMT 的机制之一。
