Background There is increasing evidence indicating an aberrant expression of miRNAs in colorectal cancer (CRC) development. Growing evidence has suggested that polyunsaturated fatty acids (PUFAs) could modulate the remodeling of the epigenome. No study has yet been published to examine the direct effect of PUFA on the promoter methylation of miRNAs. This study aimed to examine the potential clinical application of PUFA on the promoter DNA methylation of miR-126 and its angiogenic target molecule (VEGF) in the CRC cells. Methods We investigated the direct effect of 100 μM EPA, DHA, and LA for 24 h on promoter methylation status of miR-126 in a panel of five CRC cell lines (HCT116, HT29/219, Caco2, SW742, and LS180) by methylation-specific PCR (MSP). We also quantified the miR-126 and VEGF transcript expression levels in five CRC cell lines affected by PUFA by real-time PCR. Moreover, we analyzed the protein expression level of VEGF, as a target of miR-126, by western blotting assay. Results MSP analysis showed extensive DNA methylation of the miR-126 promoter in all five CRC cell lines, and among all three PUFAs, only DHA completely demethylated the promoter of miR-126 in HCT116 and Caco2 cell lines. We found that only DHA significantly induces the expression level of miR-126 in HCT116 and Caco2 cell lines, respectively, by 20.1-fold and 1.68-fold (p < 0.05). Our finding indicates that the downregulation of VEGF protein level is also effectively observed only in DHA-treated HCT116 and Caco2 cells compared to control cells (p < 0.05). Conclusions Our results provide evidence that n-3 PUFAs are able to modulate cellular miR-126 DNA methylation and inhibit VEGF expression level in a cell-type specific manner in colorectal cancer cells. DHA always showed higher efficacy than EPA and LA in our experiment. Overall, our results suggest a potential clinical application of n-3 PUFAs as anti-angiogenic agents in CRC therapy.

中文翻译：

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膳食多不饱和脂肪酸对结直肠癌细胞中 miR-126 启动子 DNA 甲基化状态和 VEGF 蛋白表达的影响。

背景 越来越多的证据表明 miRNA 在结直肠癌 (CRC) 发展中的异常表达。越来越多的证据表明，多不饱和脂肪酸 (PUFA) 可以调节表观基因组的重塑。尚未发表研究来检验 PUFA 对 miRNA 启动子甲基化的直接影响。本研究旨在探讨 PUFA 在 CRC 细胞中对 miR-126 及其血管生成靶分子 (VEGF) 的启动子 DNA 甲基化的潜在临床应用。方法 我们通过甲基化研究了 100 μM EPA、DHA 和 LA 24 小时对一组五个 CRC 细胞系（HCT116、HT29/219、Caco2、SW742 和 LS180）中 miR-126 启动子甲基化状态的直接影响-特异性PCR (MSP)。我们还通过实时 PCR 量化了 5 个受 PUFA 影响的 CRC 细胞系中的 miR-126 和 VEGF 转录物表达水平。此外，我们通过蛋白质印迹分析分析了作为 miR-126 靶标的 VEGF 的蛋白质表达水平。结果 MSP 分析显示所有 5 种 CRC 细胞系中 miR-126 启动子的 DNA 广泛甲基化，在所有 3 种 PUFA 中，只有 DHA 使 HCT116 和 Caco2 细胞系中 miR-126 的启动子完全去甲基化。我们发现只有 DHA 可显着诱导 HCT116 和 Caco2 细胞系中 miR-126 的表达水平，分别为 20.1 倍和 1.68 倍（p < 0.05）。我们的发现表明，与对照细胞相比，仅在 DHA 处理的 HCT116 和 Caco2 细胞中也有效观察到 VEGF 蛋白水平的下调（p < 0.05）。结论 我们的结果提供了证据，表明 n-3 PUFA 能够在结直肠癌细胞中以细胞类型特异性方式调节细胞 miR-126 DNA 甲基化并抑制 VEGF 表达水平。在我们的实验中，DHA 总是显示出比 EPA 和 LA 更高的功效。总体而言，我们的结果表明 n-3 PUFA 作为抗血管生成剂在 CRC 治疗中的潜在临床应用。