Analysis of cellular and exosomal circRNAs in MCF7 cells regulated by LSD1 (RNA-Seq)

Lysine-specific demethylase 1 (LSD1), a histone demethylating enzyme, plays a crucial role in cancer metastasis. While studies have demonstrated that the knockout of LSD1 significantly enhances lung metastasis of breast cancer cells, it remains unclear whether LSD1-deficient breast cancer cells can remodel the lung microenvironment to allow metastasis. In this study, we isolated exosomes from LSD1-knockdown breast cancer cells (LSD1 KD) and investigated their effects on the formation of pre-metastatic niches. The intravenous injection of exosomes from LSD1 KD cells in mice resulted in a substantial increase in lung colonization by breast cancer cells, while treatment with exosomes derived from LSD1 KD cells decreased the expression of the tight junction proteins ZO-1 and occludin in vascular endothelial cells, leading to increased pulmonary vascular permeability. In addition, the knockdown of LSD1 reduced the expression of the circular RNA circDOCK1, which augmented the levels of miR-1270 in exosomes. Further investigation showed that miR-1270 inhibited ZO-1 expression in human umbilical vein endothelial cells, which enhanced their permeability. In conclusion, our study uncovered a novel mechanism in which the epigenetic modifier LSD1 promotes the formation of pre-metastatic niches via the regulation of exosomal miRNA. Overall design: In order to find the miRNAs regulated by LSD1 in exosomes, and also to find the mechanism of miRNA regulation, we analysed circRNA (RNA-seq) expression in cells and miRNA expression in exosomes. The sequencing data from MCF7 cells were divided into 3 groups: Con, kd and Re. After analysing the differentially expressed miRNAs in the exosomes of the 3 groups of cells, the circRNAs were also differentially analysed to explore the corresponding mechanisms.

赖氨酸特异性去甲基化酶1（Lysine-specific demethylase 1, LSD1）是一种组蛋白去甲基化酶，在肿瘤转移过程中发挥关键调控作用。已有研究表明，敲除LSD1可显著增强乳腺癌细胞的肺转移能力，但目前仍未明确LSD1缺陷型乳腺癌细胞是否能够重塑肺微环境以促进肿瘤转移。本研究从LSD1敲低（LSD1 KD）乳腺癌细胞中分离外泌体（exosomes），并探究其对转移前微环境形成的影响。向小鼠静脉注射LSD1 KD细胞来源的外泌体后，乳腺癌细胞的肺定植能力显著提升；而经LSD1 KD细胞外泌体处理的血管内皮细胞，其紧密连接蛋白ZO-1与闭合蛋白（occludin）的表达水平均出现下调，进而导致肺血管通透性升高。此外，LSD1敲低可降低环状RNA circDOCK1的表达，这一变化会使外泌体中miR-1270的水平显著升高。进一步研究显示，miR-1270可抑制人脐静脉内皮细胞（human umbilical vein endothelial cells）中ZO-1的表达，从而增强其细胞通透性。综上，本研究揭示了一种全新的分子机制：表观遗传修饰因子LSD1可通过调控外泌体miRNA的表达，促进肿瘤转移前微环境的形成。整体实验设计：为筛选外泌体中受LSD1调控的miRNA并阐明其调控机制，本研究对细胞内环状RNA（circRNA，经RNA-seq分析）的表达水平以及外泌体中的miRNA表达水平进行了检测。来自MCF7细胞的测序数据被分为Con组、kd组与Re三组，在完成三组细胞外泌体的差异表达miRNA分析后，本研究同时对circRNA进行差异表达分析，以探究对应的分子调控机制。