Wang Wei, Zhang Yuefen, Huang Xiaomin, Li Dan, Lin Qi, Zhuang Hailin, Li Hong
School of Public Health and Health Management, Fujian Health College, Fuzhou, Fujian, China.
Science and Technology Service Center, Fujian Health College, Fuzhou, Fujian, China.
Front Pharmacol. 2024 Aug 22;15:1445034. doi: 10.3389/fphar.2024.1445034. eCollection 2024.
Rosmarinic acid (RA), a natural phenolic acid, exhibits promising anti-cancer properties. The abnormal expression of microRNA (miRNA) regulates the gene expression and plays a role as an oncogenic or tumor suppressor in TNBC. However, the biological role of RA in miR-30a-5p on BCL2L11 during MDA-MB-231 induced breast cancer stem-like cells (BCSCs) progression and its regulatory mechanism have not been elucidated.
To investigate whether RA inhibited the silencing effect of miR-30a-5p on the BCL2L11 gene and promoted apoptosis in BCSCs.
We assessed the migration, colony formation, proliferation, cell cycle, and apoptosis of BCSCs after RA treatment using the wound-healing assay, colony formation assay, CCK-8 assay, and flow cytometry, respectively. The expression of mRNA and protein levels of BCL-2, Bax, BCL2L11, and P53 genes in BCSCs after RA treatment was obtained by real-time polymerase chain reaction and Western blot. Differential miRNA expression in BCSCs was analyzed by high-throughput sequencing. Targetscan was utilized to predict the targets of miR-30a-5p. The dual luciferase reporter system was used for validation of the miR-30a-5p target.
Wound-healing assay, colony formation assay, CCK-8 assay, and cell cycle assay results showed that RA inhibited migration, colony formation and viability of BCSCs, and cell cycle arrest in the G0-G1 phase. At the highest dose of RA, we noticed cell atrophy, while the arrest rate at 100 μg/mL RA surpassed that at 200 μg/mL RA. Apoptotic cells appeared early (Membrane Associated Protein V FITC, PI) or late (Membrane Associated Protein V FITC, PI) upon administration of 200 μg/mL RA, Using high-throughput sequencing to compare the differences in miRNA expression, we detected downregulation of miR-30a-5p expression, and the results of dual luciferase reporter gene analysis indicated that BCL2L11 was a direct target of miR-30a-5p.
RA inhibited the silencing effect of miR-30a-5p on the BCL2L11 gene and enhanced apoptosis in BCSCs.
迷迭香酸（RA）是一种天然酚酸，具有良好的抗癌特性。微小RNA（miRNA）的异常表达可调节基因表达，并在三阴性乳腺癌（TNBC）中发挥致癌或抑癌作用。然而，RA在miR-30a-5p对BCL2L11的作用中，在MDA-MB-231诱导的乳腺癌干细胞（BCSCs）进展过程中的生物学作用及其调控机制尚未阐明。
研究RA是否能抑制miR-30a-5p对BCL2L11基因的沉默作用，并促进BCSCs凋亡。
我们分别采用划痕实验、集落形成实验、CCK-8实验和流式细胞术评估RA处理后BCSCs的迁移、集落形成、增殖、细胞周期和凋亡情况。通过实时聚合酶链反应和蛋白质免疫印迹法检测RA处理后BCSCs中BCL-2、Bax、BCL2L11和P53基因的mRNA和蛋白质水平表达。通过高通量测序分析BCSCs中差异miRNA表达。利用Targetscan预测miR-30a-5p的靶标。采用双荧光素酶报告系统验证miR-30a-5p靶标。
划痕实验、集落形成实验、CCK-8实验和细胞周期实验结果表明，RA抑制了BCSCs的迁移、集落形成和活力，并使细胞周期停滞在G0-G1期。在RA最高剂量时，我们注意到细胞萎缩，而100μg/mL RA时的停滞率超过200μg/mL RA时的停滞率。给予200μg/mL RA后，早期（膜联蛋白V FITC、PI）或晚期（膜联蛋白V FITC、PI）出现凋亡细胞。利用高通量测序比较miRNA表达差异，我们检测到miR-30a-5p表达下调，双荧光素酶报告基因分析结果表明BCL2L11是miR-30a-5p的直接靶标。
RA抑制了miR-30a-5p对BCL2L11基因的沉默作用，并增强了BCSCs的凋亡。
