Department of Preclinical Medicine, Medical College, Hunan Normal University, Changsha 410013, China.
Department of Preclinical Medicine, Medical College, Hunan Normal University, Changsha 410013, China.
Toxicol Appl Pharmacol. 2020 Jun 1;396:114998. doi: 10.1016/j.taap.2020.114998. Epub 2020 Apr 5.
Casticin (CAS) is a polymethyl flavonoid from Fructus viticis and has multiple pharmacological activities, including anticancer. However, whether the molecular mechanism underlying CAS represses stemness characteristics in hepatocellular carcinoma (HCC) cells involves intervention in the reciprocal negative regulation between DNA methyltransferase 1 (DNMT1) and miR-148a-3p has not yet been reported. In this study, the effect of CAS on stemness characteristics of HCC cells and its mechanism were investigated. Results showed that CAS selectively reduced the viabilities of HCC cells but not L02 cells, as determined by CCK-8 assay. Importantly, the sub-cytotoxic concentrations of CAS could inhibit the stemness characteristics in HCC cells, as demonstrated by the expression of stemness biomarkers (CD44, EpCAM, Bmi1, Nanog, and Oct4), sphere forming assay, RT-qPCR, and Western blotting. In addition, CAS repressed DNMT1 activity and expression and increased miR-148a-3p. The effect of CAS on stemness characteristics was abolished by stable DNMT1 overexpression. MiR-148a-3p overexpression enhanced the reduction of CAS on stemness characteristics. DNMT1 overexpression promoted miR-148a-3p promoter hypermethylation as detected by methylation-specific PCR (MSP), which repressed its expression. Conversely, miR-148a-3p repressed DNMT1 expression by specific site binding to 3'-UTR of DNMT1 mRNA, as determined by luciferase assay. Moreover, the combination of CAS and agomir-148a-3p had robust effects on tumor suppression as compared to the sole activity of either molecule in nude mouse xenograft experiments in vivo. The findings suggested that CAS could inhibit stemness characteristics in HCC cells by interruption of the reciprocal negative regulation between DNMT1 and miR-148a-3p.
金合欢素（CAS）是牡荆素，来源于葡萄科，具有多种药理活性，包括抗癌作用。然而，金合欢素（CAS）是否通过干预 DNA 甲基转移酶 1（DNMT1）和 miR-148a-3p 的相互负调控来抑制肝癌（HCC）细胞的干性特征，其分子机制尚未报道。在本研究中，研究了 CAS 对 HCC 细胞干性特征的影响及其机制。结果表明，通过 CCK-8 测定，CAS 选择性降低 HCC 细胞但不降低 L02 细胞的活力。重要的是，亚细胞毒性浓度的 CAS 可抑制 HCC 细胞的干性特征，表现在干细胞标志物（CD44、EpCAM、Bmi1、Nanog 和 Oct4）的表达、球体形成试验、RT-qPCR 和 Western blot 中。此外，CAS 抑制了 DNMT1 活性和表达，增加了 miR-148a-3p 的表达。稳定过表达 DNMT1 可消除 CAS 对干性特征的影响。miR-148a-3p 的过表达增强了 CAS 对干性特征的降低作用。DNMT1 过表达促进了 miR-148a-3p 启动子的超甲基化，如甲基特异性 PCR（MSP）所检测到的，从而抑制其表达。相反，miR-148a-3p 通过特异性结合到 DNMT1 mRNA 的 3'UTR 抑制了 DNMT1 的表达，如通过荧光素酶测定所确定的。此外，与单独使用 CAS 或 agomir-148a-3p 相比，在体内裸鼠异种移植实验中，CAS 和 agomir-148a-3p 的联合使用对肿瘤抑制具有更强的作用。这些发现表明，CAS 通过中断 DNMT1 和 miR-148a-3p 之间的相互负调控，抑制 HCC 细胞的干性特征。
